1.
Gene Therapy
Balasubramanian Thiagarajan
drtbalu's otolaryngology online

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.
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

6.
Types of vectors used in gene therapy
 Replication deficient viruses
 Liposomes
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

8.
Gene therapy models for cystic fibrosis
 Nasal model
 Pulmonary model
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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.
drtbalu's otolaryngology online

11.
Viral vectors
 Adenovirus
 Adeno associated virus
 Lentivirus
drtbalu's otolaryngology online

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.
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

17.
Head & Neck Malignancy
And gene therapy
drtbalu's otolaryngology online

18.
Role of gene therapy in Head & Neck Ca
 Immune modulation
 Restorative gene replacement
 Selective oncolysis
 Chemosesitization
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

27.
Gene therapy strategies
 Chemosensitization
 Cytokine gene transfer
 Inactivation of protooncogene production
 Selective oncolysis
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

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
drtbalu's otolaryngology online

32.
Thank you
drtbalu's otolaryngology online