gene therapy otolaryngology

Gene TherapyBalasubramanian Thiagarajan

drtbalu's otolaryngology online

Introduction

1. Genome influences all aspects of human life

2. Each human cell contains 23 pairs of chromosomes (one pair is from maternal and the other from paternal sources)

3. Human genome has been broken down with reasonable degree of accuracy by the Human genome project.

4. Gene is a functional unit which serves as a template for production of proteins by the cell

5. Genes are present in a genome as a single contiguous sequence of DNA, but often they are broken down into smaller stretches known as exons which are separated by stretches of non coding DNA known as introns.

6. Messenger RNA copies the DNA templates from genes. After this transcription process the introns are removed from the copy by splicing.


Alternate splicing

Some messenger RNA copies can be made with some exons excluded.

Some messenger RNA copies can be made with exons used in different

combinations

This method is responsible for production of wide variety of molecules from a

small number of 30,000 genes


Polymorphism

DNA sequence from different individuals differs at the rate of one base /

thousand. This variation is known as polymorphism.

Substitution of one base by the another

Deletion / addition of one or more bases on large DNA stretches

Polymorphism influences the development and function of an individual.

The term Mutation has been coined to indicate polymorphism with

deleterious effects.

Polymorphism can also have beneficial effects

Modifiers are variants of genes that can either ameliorate / exacerbate the

effects of a single gene mutation


Gene as therapeutic tool

63 human trials are currently going on using gene as therapy

Genetic material can be delivered to target cells using vectors to bypass the

immune mechanism. This aims to repair / replace the disease causing gene.

3 groups of disorders are currently being evaluated for gene therapy: they

include:

Infectious disease trials – (HIV)

Monogenetic diseases – Cystic fibrosis & Hemophilia B

Polygenetic diseases – Rheumatoid arthritis / Cancer


Types of vectors used in gene therapy

Replication deficient viruses

Liposomes


Cystic Fibrosis

Single mutation & autosomal recessive inheritance

Caused due to mutations involving CF gene in the long arm of chromosome 7

CF gene encodes a protein known as cystic fibrosis transmembrane

conductance regulator (CFTR protein).

Commonest lethal genetic disorder involving the wealthiest racial groups

(United States of America) hence research funding is never a problem


Gene therapy models for cystic fibrosis

Nasal model

Pulmonary model


Nasal model of gene therapy

Promising in cystic fibrosis

Cystic fibrosis has nasal manifestations

Gene administration by virus vectors are safer via nasal cavity

Nasal and sinus mucosa have fairly large surface area adequate for

absorption of the vector.

Adenovirus which is commonly used as vector has a propensity to adsorb to

nasal mucosa

Treatment efficacy can easily be ascertained by measuring the potential

difference across the nasal mucosa which is easy to perform


Pulmonary model

Life threatening complication of cystic fibrosis is due to its effect on the

lungs

Adenovirus which is the commonly used vector for transportation of genetic

material easily adheres to the alveolar mucosa

This virus has deleterious effects on the lung tissue causing death also, hence

caution is to be exercised while this model is preferred.


Viral vectors

Adenovirus

Adeno associated virus

Lentivirus


Adenovirus vectors

These viruses have a tropism for respiratory mucosa

Recombinant adenovirus is prepared by replacing DNA sequence responsible

for replication of virus with that of DNA sequence responsible for secretion of

CFTR protein which is deficient in patients with cystic fibrosis

Human respiratory mucosa has sufficient immunity to prevent adenovirus

infections. Hence administration of genes using this virus via pulmonary route

may not be effective

Nasal route is ideally suited for gene administration via adenovirus in

patients with cystic fibrosis.


Role of calcium chelators in adenovirus

gene therapy

Adenovirus vectors are aimed at epithelial surface

In pulmonary epithelium viral receptors are located in the basolateral

membrane away from the surface

Integration of these viral vectors with calcium chelators will cause transient

disruption of tight epithelial junctions allowing vector access to the

basolateral membrane


Adeno associated virus

Serotypes 5&6 of AAV enters airway cells from the apical surface

This virus offers scope of potential integration into the host genome

Direct administration into maxillary sinuses have been attempted with

reasonable success

Administration into maxillary sinuses do not cause sinusitis

Measurement of maxillary sinus voltage helps in the determination of

therapeutic end point


Lentivirus

Feline immunodeficiency virus is an example

They can integrate and persist in the host genome

They can transduce into non dividing cells. This is helpful in the pulmonary

mucosa whose turnover is rather low


Non viral vectors

Purified / naked DNA in plasmid form is used

Gene gun / ballistic delivery system – can be used only on exposed surfaces

Liposmes – holds lots of promise – DNA coated liposomes gets incorporated

into the cell by endocytosis

These non viral vectors are non immunogenic

Flip side – gene transfer is inefficient


Head & Neck Malignancy

And gene therapy


Role of gene therapy in Head & Neck Ca

Immune modulation

Restorative gene replacement

Selective oncolysis

Chemosesitization


Ideal gene delivery system

To achieve expression of gene of interest in the targeted cancer cell

Malignant cells need to be targeted

Binding and internalization of genes by the targeted cells

Gene should escape endosomal degradation and reach the nucleus

Nuclear expression – once inside the nucleus the quantity of gene expression

should be adequate and stable


Plasmid DNA - Advantages

Also known as naked DNA

Non viral method of gene transmission

Simple in concept

Easily mass produced

Minimal immune response

Relatively safer when compared to viral delivery systems


Plasmid DNA - Disadvantages

Least efficient

Majority of DNA not internalized

Even if internalized endosomal degradation almost always destroys it

Since it is not receptor mediated accurate targeting is nearly impossible


Efficacy of plasmid DNA – How to

improve?

Complexing DNA with lipids / peptides / polymers /cations have increased the

chances of expression. These methods improve internalization and cell

binding.

Linking DNA to a ligand helps in targeting specific tissues

Plasmid mediated expression is only transient because plasmid is lost after

cell division. This is a major stumbling block which is yet to be resolved


Adenovirus as a vector

Efficiently infects both dividing and non dividing cells because of its ability to

bind to cox-adenovirus receptor

Internalization and trafficking to the nucleus is efficient

It can be combined with complexes that hastens internalization

Only draw back being its immunogenicity which prevents it from reinfecting

cells. Both humoral and CMI are activated after delivery of virus


Reducing immunogenicity of adenovirus

Gutted version of the virus can be prepared by removing all viral genes

thereby reducing its immunogenicity

Immune modulation can be attempted at the time of delivery reducing

inflammation


Role of retroviral vectors

These are small encapsulated RNA viruses

Major advantage of this vector is persistent gene expression

In these vectors the viral genes that causes virus to replicate has been

removed to make it non replicative

Murine oncogenic retroviruses are being currently tested for this purpose


AAV-Adeno associated vectors

Single stranded encapsulated virus – Parvovirus family

This virus does not cause pathologic disease in humans

It causes very little immunogenicity

It persists and infects dividing and non dividing cells


Gene therapy strategies

Chemosensitization

Cytokine gene transfer

Inactivation of protooncogene production

Selective oncolysis


Chemo sensitization

Selective sensitization of cancer cells to chemo RT

Ideal way to kill cancer cells

Gene delivery should be targeted i.e. only malignant cells should receive the

genes

Herpes simplex thymidine kinase can be delivered to cancer cells making

them more susceptible to Gancyclovir

Bystander effect ensures that the cancer cells spread these genes into cells

surrounding them via cell to cell contact

Transfer of p53 gene sensitizes the cancer cell to undergo apoptosis following

chemotherapy / irradiation


Cytokine gene transfer

Immune dysfunction at the site of tumor causes malignant cells to thrive.

Immunological ignorance / down regulation of major histocompatibility

complexes have been attributed

Over expression of down regulated cytokines could help. This is where gene

therapy comes in since direct administration of cytokines have proven to be

toxic due to capillary leak syndrome


Restorative gene therapy

Mutations of p 53 and p16 genes are common in sq cell carcinoma of head and

neck. Restoration of these mutated genes could enhance apoptosis of tumor

cells

This can also inactivate proto oncogene production

Repair of deranged apoptotic pathway in tumor cells could help in controlling

malignancy


Selective oncolytic viruses

Infections with wild type adenovirus can cause excessive replication of the

viruses leading on to cell death

If these viruses could be harnessed to replicate inside cancer cells alone then

preferential targeted destruction of tumor cells becomes a possibility

ONYX – 015 is a adenovirus specifically designed to replicated inside tumor

cells that lack functional p 53 gene. Nearly 60% of head and neck sq

carcinoma tumor cells lack functional p 53 gene

If administered intravenously it can take care of distant metastasis


Thank you


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