In The Name Of God

Targeting Gene Therapy Targeting Gene Therapy to Cancerto Cancer

Abstract

In recent years the idea of using gene therapy specially,cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression,specific gene product activity and specific drug activation.

This presentation will discuss the progress has made in recent years about the efficient delivery of DNA to tumour sites.

IntroductionIntroduction

MAJOR MAJOR DEVELOPMENT IN DEVELOPMENT IN GENE THERAPYGENE THERAPY

19891989::THE FIRST PROTOCOL FOR THE FIRST PROTOCOL FOR CANCER GENE THERAPYCANCER GENE THERAPY

19901990::GENE THERAPY TO TREAT SCID GENE THERAPY TO TREAT SCID USING STEM CELLSUSING STEM CELLS

19931993::NEW GENE THERAPY APPROACH NEW GENE THERAPY APPROACH REPAIRS ERRORS IN mRNAREPAIRS ERRORS IN mRNA

New approach using liposome 25 New approach using liposome 25 nanometersnanometers

SICKLE CELL DISEASE IS A SUCCESSFULLY TREATED IN MICE

2003:USING LIPOSOME COATED WITH PEG TO TREAT PARKINSON DISEASE

USING siRNAs TO TREAT HUNTINGTON

2006:THE FIRST REPORT THAT GENE THERAPY CAN BE EFFECTIVE IN TREATING CANCER

PREVENTION THE IMMUNE SYSTEM WITHMicro RNA

2007:THE FIRST GENE THERAPY TRIAL FOR INHERITED RETINAL DISEASE

2008:TREATMENT OF LEBER CONGENITAL DISEASE

TYPE OF GENE THERAPY

SOMATIC CELL GENE THERAPY

GERM LINE GENE THERAPY

GENE THERAPY

***REMOVE GENE FROM A SELECTED TISSUE

***EXPOSITION TO THE GENE TRANFER VECTOR

***SELECTION FOR TRANSGENIC USING MARKER

***REINTRODUCTION THE CORRECTED IN TO THE PATIENT BODY

GENE THERAPYGENE THERAPY

INJECTION THE VECTOR DIRECTLY INJECTION THE VECTOR DIRECTLY IN TO THE TARGETED TISSUEIN TO THE TARGETED TISSUE

BROAD METHODSBROAD METHODS

******REPLACE A NON FUNCTIONAL GENEREPLACE A NON FUNCTIONAL GENE

******HOMOLOGOUS RECOMBINATIONHOMOLOGOUS RECOMBINATION

******SELECTIVE REVERSE MUTATIONSELECTIVE REVERSE MUTATION

******THE ALTERATION OF THE GENE THE ALTERATION OF THE GENE REGULATIONREGULATION

To increase specificity and safety of gene therapy needs to:

Gene delivery using viral and non-viral method.

Targeting gene expression

Condition targeted expression

Disease targeted expression

Targeted delivery

Molecular

chemotherapy

Genetic

immunopotentiationMutation

compensation

Delivery of suicide gene

Gene directed enzymeProdrug therapy

Viral and

non-viral

.1methods

Viral-mediated

methods

Lipid-mediated

methods

The

others

retrovirus HVJAdeno

virusLiposome

Micro

injection

Microparticle

bombardment

LIPOLEXES

ANIONIC NEUTRAL CATIONIC

POLYLEXES

CHITOSANTRIMETHL CHITOSAN

HYBRID METHODSHYBRID METHODS

VIROSOMESVIROSOMES((LIPOSOME +INACTIVATED HIVLIPOSOME +INACTIVATED HIV))

DENDRIMERS

Non solid tumoursNon solid tumours::

Herpes simplex virus thymidine Herpes simplex virus thymidine kinase and cytosine deaminasekinase and cytosine deaminase

Modified envelope proteinsModified envelope proteins

Targeted delivery of DNA via Targeted delivery of DNA via receptorreceptor

Solid tumoursSolid tumours::An aerobic bacteriaAn aerobic bacteria

Retroviral vectors with modified envelope proteins

Mo-MuLV

SU TM

Binding ofthe virus to

Its cell receptor

Anchors the molecule to

the viral membrane

SPECIFIC TARGETING STRATEGIES OF CANCER

GENE THERAPY USING scFv

•RETROVIRAL VECTOR DISPLAYS BOTH ANTI CEA scFv AND Inos gene

•CHIMARIC RECEPTOR GENE WHICH ENCODED AN NATI CEA scFv AND THE ZETA CHAIN OF TCR/CD3

Delivery of suicide geneDelivery of suicide gene

Targeted delivery of Targeted delivery of DNA via receptorDNA via receptor

•REDIRECTING VIRUS TO A TISSUE REDIRECTING VIRUS TO A TISSUE SPECIFIC RECEPTORSPECIFIC RECEPTOR

•USING TISSUE SPECIFIC LIGANDS OR USING TISSUE SPECIFIC LIGANDS OR mAb INCORPORATED ON TO THE mAb INCORPORATED ON TO THE SURFACE OF LIPOSOME TO DIRECT SURFACE OF LIPOSOME TO DIRECT THEM TO TARGET CELLSTHEM TO TARGET CELLS

Targeted delivery and receptor overexpressing human cells

EGF-R

Folate-R

C-kit-R

VDEPTVDEPT

Virally directed enzyme Virally directed enzyme prodrug therapyprodrug therapy

SYNTHETIC OLIGONUCLEOTIDES

ANTISENSE siRNA

DOUBLE STRANDED OLIGODEOXYNUCLEOTIDES

PEPTIDE NUCLEIC ACID SEQUENCE SPECIFIC

RECOGNITION IN TARGETING GENE THERAPY TO CANCER

PNA IN CANCER GENE THERAPY

•***INHIBITION OF TELOMERASE ACTIVITY

•***ANTISENSE AND ANTIGENE ACTIVITY OF PNA TO HUMAN B-CELL LYNPHOMA

•***INHIBITION OF HIV REVERSE TRANSCRIPTASE

•***PNA AS A GENETIC ANTIBIOTICS

•***DIHYDROTESTOSTRONE LINKED TO PNA AS A VECTOR FOR TARGETING C-MYC DNA TO PROSTATIC CANCER CELLS

EFFECTIVE METHODS TO EFFECTIVE METHODS TO INDUCE UPTAKE OF PNA IN TO INDUCE UPTAKE OF PNA IN TO CELLSCELLS

******ELECTROPORATIONELECTROPORATION

******CATIONIC LIPIDSCATIONIC LIPIDS

******ADENO VIRUS-POLYLYSINE ADENO VIRUS-POLYLYSINE COMPLEXESCOMPLEXES

******STEREPTOLYSINESTEREPTOLYSINE

TRANSCRIPTIONAL CONTROL OF EXPRESSION

T0MOUR SPECIFIC PROMOTERST0MOUR SPECIFIC PROMOTERS

11))PROMOTERS THAT SPECIFIC ONLY PROMOTERS THAT SPECIFIC ONLY IN MALIGNANT PROCESSIN MALIGNANT PROCESS

22))PROMOTERS THAT ONCOFOETAL PROMOTERS THAT ONCOFOETAL RELATED WITH TISSUE SPECIFICITYRELATED WITH TISSUE SPECIFICITY..

33))TOMOUR MICROENVIROMENT-TOMOUR MICROENVIROMENT-RELATED PROMOTERSRELATED PROMOTERS

44))TOMOUR VASCULATURE-RELATED TOMOUR VASCULATURE-RELATED PROMOTERSPROMOTERS..

Tissue targeted expression

Alpha feto protein to Alpha feto protein to target hepatocellular target hepatocellular

carcinomacarcinoma

AFP has been found to be abnormally activated in hepatocellular carcinoma.

Transgenic mice carrying the SV40 large T-antigen developed carcinoma crossed with mice transgenic for HSV thymidine kinase.

HSVtk activate GCV under the AFP promoter/enhancer control.

•Tissue targeted expressionTissue targeted expression•Albumin enhancer to target liver Albumin enhancer to target liver

cancercancer

Retroviral vector containing the liver-specific expression system.

The albumin enhancer element and promoter to target expression of B-gal to hepatoma cells.

Expression could only be detected in dividing hepatocytes.

Condition targeted expression

Tissue-type plasminogen activator regulated Tissue-type plasminogen activator regulated by radiationby radiation..

t-pA induces over 50 fold after irradiation t-pA induces over 50 fold after irradiation with x-rayswith x-rays..

The t-pA protease have a function The t-pA protease have a function equivalent to the sos repair systemequivalent to the sos repair system..

GRP encoding gene was used to control expression of a marker gene in a murine fibrosarcoma model.

In vitro glucose deprivation of transduced fibrosarcoma cells showed an 8-fold induction over non-stressed cells.

Hypoxia regulated gene expressionHypoxia regulated gene expression)in almost solid tumours()in almost solid tumours(

Hypoxic conditions can modulate Hypoxic conditions can modulate the expression of a number genesthe expression of a number genes::

Encoding growth factorsEncoding growth factors..

Transcription factorsTranscription factors..

Glycolytic and DNA repair enzymesGlycolytic and DNA repair enzymes..

Hypoxic expression is controlled by the Hypoxic expression is controlled by the binding of transcription factor(HIF-1) to a binding of transcription factor(HIF-1) to a DNA sequence which can be enhancer or DNA sequence which can be enhancer or response element.(HRE)response element.(HRE)

The use of hypoxia enhancer elements increase the levels of the marker gene CD2.

Hypoxic induction in vivo of the CD2 was confirmed by CD2 staining with the comet assay.

In comet assay,tumour cells are treated with radiation and a bioreductive drug)which indices DNA crosslinks only in hypoxic cells(

Electrophoresis of single cells isolated from the treated tumours can differentiate the two populations.

The hypoxic cells,according to the comet The hypoxic cells,according to the comet assay,stained positive for CD2,whereas the assay,stained positive for CD2,whereas the aerobic ones did notaerobic ones did not..

This result demonstrate the selectivity of the This result demonstrate the selectivity of the system and its potential for tumour specific system and its potential for tumour specific targeting of gene expressiontargeting of gene expression..

INDUCTION OF APOPTOSIS IN INDUCTION OF APOPTOSIS IN HYPOXIC CONDITIONHYPOXIC CONDITION

P53P53

BAXBAX

hREC2hREC2

Caspase-8Caspase-8

Genetically modified tomour vaccines in gene therapy

Cytokine genes

Co-stimulatory moleculesCo-stimulatory molecules

DNA VACCINE

ConclusionConclusion

Gene therapy has the potential to have few side effects and lower systemic toxicity than current therapies.

Also choosing only one criteria for selectivity such as targeting delivery to cells or tissue specific expression,is not sufficient.

Only by combining the Only by combining the most successful strategies most successful strategies in cancer gene therapy in cancer gene therapy approaches,will be a approaches,will be a successful clinical successful clinical treatment emergetreatment emerge..