gene therpy

GENE THERAPY : 

GENE THERAPY

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Introduction Strategy Approach Types Limitation Gene therapy in cancer Various clinical application Future aspects

Introduction : 

Introduction Gene therapy (G T) To restore missing function Amplify existing functions To impart the new function to the cell

Strategy : 

Strategy Gene augmentation therapy i.e. replacing the defective gene by functional gene Gene inhibition therapy i.e. inserting the anti sense gene which inhibits the expression of dominant gene

Approaches : 

Approaches Somatic cell gene therapy ex: bone marrow cell blood cell skin cell intestinal cell At present most of the GT are directed towards Somatic cell GT

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Germ cell G T Ex: reproductive cells The parent’s egg or sperm cells are changed with the objective of passing on the changes to the offspring.

Types : 

Types

Example of ex vivo GT : 

Example of ex vivo GT Usually done with blood cells because they are easiest to remove and return. Sickle cell anemia

Example of in situ somatic cellgene therapy : 

Example of in situ somatic cellgene therapy • Infusion of adenoviral vectors into the trachea and bronchi of cystic fibrosis patients. • Injection of a tumor mass with a vector carrying the gene for a cytokine or toxin. • Injection of a dystrophin gene directly into the muscle of muscular dystrophy patients.

Example of in-vivo somatic cellgene therapy : 

Example of in-vivo somatic cellgene therapy • No clinical examples. • In vivo injectable vectors must be developed

Ex vivo manipulation techniques : 

Ex vivo manipulation techniques Electroporation Liposome Calcium phosphate Gold bullets (fired within helium pressurized gun) Retrotransposons (jumping genes – early days) Human artificial chromosomes

Electro poration : 

Electro poration

Liposome : 

Liposome

Gene guns : 

Gene guns Isolate normal DNA fragment. Make very small spheres of a heavy metal like gold. Coat gold with DNA. Fire particles at cells at high speed so particles enter cells. May be used directly on tissues or organs in situ

In vivo techniques usually utilize viral vectors : 

In vivo techniques usually utilize viral vectors Virus = carrier of desired gene Virus is usually “crippled” to disable its ability to cause disease Viral methods have proved to be the most efficient to date Many viral vectors can stable integrate the desired gene into the target cell’s genome

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Ideal vector characteristics: Insert size: one or more genes. Targeted: limited to a cell type. No immune response. Stable: not mutated. Production: easy to produce high concentrations 6. Regulatable: produce enough protein to cause an effect.

Types of vectors : 

Types of vectors • RNA viruses (Retroviruses) 1. Murine leukemia virus (MuLV) 2. Human immunodeficiency viruses (HIV) 3. Human T-cell lymphotropic viruses (HTLV) • DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses Non-viral vectors 1. Liposomes 2. Naked DNA 3. Liposome-polycation complexes 4. Peptide delivery systems

Adeno virus : 

Adeno virus

Why Viruses? : 

Why Viruses? Viruses through the time of evolution have evolved to infect the cells with great specificity Viruses tend to be very efficient at transfecting their own DNA into the host cell genome. This allows them to produce new viral particles at the period of synthesis of the cell

Which Virus to Use? : 

Which Virus to Use? Depends on how well they transfer the genes to cells which cells they can recognize and infect and whether they alter the cell’s DNA permanently or temporarily

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Human artificial chromosome 47th artificial chromosome to the body. It would exist autonomously along side of the other 46 chromosomes, not affecting their workings or causing any mutations. large vector No immune response

Ethical Considerations : 

Ethical Considerations Use of technology for non-disease conditions such as functional enhancement or “cosmetic” purposes In utero somatic gene therapy Potential for real abuse exists by combining cloning and genetic engineering

Limitations of Gene Therapy : 

Limitations of Gene Therapy Gene delivery Limited tropism of viral vectors Dependence on cell cycle by some viral vectors (i.e. mitosis required) Duration of gene activity Non-integrating delivery will be transient (transient expression) Integrated delivery will be stable

Limitations of Gene Therapy : 

Limitations of Gene Therapy Patient safety -Immune hyper responsiveness -Integration if not controlled ? oncogenes may be involved at insertion point ? cancer Expense

Limitations of Gene Therapy : 

Limitations of Gene Therapy Controlled gene expression

Gene therapy in cancer : 

Gene therapy in cancer

Suicide gene therapy : 

Suicide gene therapy Involves the transfer of genes coding Thymidine kinase Cytosine de-aminase Examples : Brain tumor Breast tumor Prostate tumor

Suppressor gene therapy : 

Suppressor gene therapy Several genes have been discovered that are important to cancer origins and also control the cell cycle Examples : RB P53 mdm2 Cip1 P16 Cyclin D

P 53 : 

P 53 Role of P53 is to destroy cells with damaged DNA It is a transcription factor that triggers cell cycle arrest and cell death P53 is altered and defective in about 50 % of all lung, breast, and colorectal cancers Replace the defective gene by functional gene

Oncolytic virus : 

Oncolytic virus

Chemo modulation : 

Chemo modulation Aim : tissue protection MDR-1 gene is translated to hemopoietic cells Code for CM – drug efflux pump Thus, BM toxicity is decreased and can use more intense and aggressive protocols

Immuno modulation : 

Immuno modulation Turns on Immune System vs. cancer Remove cancer cells from body Insert genes that make cytokines IL-2, IL-7, TNF-a, GM-CSF Return reengineered cells to body; they attract immune system to kill cancer.

Anti angiogenesis : 

Anti angiogenesis ? the expression of angiogenesis inhibitors ex. Angiostatin, endostatin or ? the levels of VEGF / VERF-R or Combined approach

Future aspects of gene therapy : 

Future aspects of gene therapy RNA interference Exon skipping Anti sense therapy

RNA interference : 

RNA interference D/s RNA similar to the gene targeted to suppression Si RNA / Sh RNA (Hair pin RNA) Enzymatic degradation of the target gene Prevent the translation of the protein

RNA interference : 

RNA interference

Exon skipping : 

Exon skipping In Duchene muscular dystrophy – frame shift mutation of the gene dystrophin Short sequence of RNA is inserted Prevents the splicing of introns and exons Results in Becker muscular dystrophy - frame mutation of the gene dystrophin

Exon skipping : 

Exon skipping

Anti sense therapy : 

Anti sense therapy Oncogenes ? cancer Target these genes by anti sense genes or oligonucleotides Two ways -Anti sense c DNA -Anti sense m RNA Ex. -In Glioma – IGF-1 -In Prostate ca – IGF-1R

Anti sense therapy : 

Anti sense therapy

SCID : 

SCID Due to deficiency of adenosine de aminase Gene located on chr.22 Deficiency results in failure to develop functional T and B lymphocytes

SCID : 

SCID ADA is involved in purine degradation Accumulation of nucleotide metabolites = TOXIC to developing T lymphocytes B cells don’t mature because they require T cell help Patients cannot withstand infection ? die if untreated

SCID : 

SCID