2. 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.
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3. 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
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4. 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
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5. 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
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6. Types of vectors used in gene therapy
Replication deficient viruses
Liposomes
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7. 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
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8. Gene therapy models for cystic fibrosis
Nasal model
Pulmonary model
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9. 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
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10. 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.
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12. 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.
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13. 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
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14. 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
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15. 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
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16. 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
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17. Head & Neck Malignancy
And gene therapy
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18. Role of gene therapy in Head & Neck Ca
Immune modulation
Restorative gene replacement
Selective oncolysis
Chemosesitization
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19. 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
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20. 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
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21. 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
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22. 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
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23. 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
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24. 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
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25. 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
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26. 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
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27. Gene therapy strategies
Chemosensitization
Cytokine gene transfer
Inactivation of protooncogene production
Selective oncolysis
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28. 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
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29. 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
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30. 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
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31. 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
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