Dev gene therapy

BY

B.DHARMA DEV(10T22S0105)

MENTOR

Ms.P.VEDAVATHI

According to the official guidelines for human nomenclature, a gene is defined as a DNA segment that contributes to phenotype/ function.

1969- first gene is isolated.1970- first artificial gene was made

Introduction of normal genes into cells that contain defective genes to reconstitute a missing protein product.

GT is used to correct a deficient phenotype so that sufficient amounts of normal gene products are synthesized.

Transcription

Translation

Therapeutic protein

Vector for efficient gene delivery

Therapeutic gene (Transgene)

How to deliver genes to specific cells and tissues and whole animals?

How much and how long the introduced gene will be expressed.

The site and dose of gene delivery.

Any toxic effects.

Is there any adverse immunological consequence of both vehicle and gene ??

Germ cell line therapy

Somatic gene therapy

Ex vivo

In vivo

In situ

VIRAL VECTORS NON-VIRAL VECTORS

DNA viruses Adenovirus Adeno associated

virus Herpes simplex virus Vaccinia virus

RNA viruses Retroviruses

In Vitro methods: Calcium phosphate

transfection. Micro injection. Cell electroporation.

In vitro and In vivo methods:

Liposomes Naked plasma DNA

injection. Ballistic DNA

injection.

☻High concentration of virus allowing many cells to be infected High concentration of virus allowing many cells to be infected

or transducedor transduced

☻Convenience and reproducibility of productionConvenience and reproducibility of production

☻Ability to transduce dividing and non-dividing cellsAbility to transduce dividing and non-dividing cells

☻Ability to integrate into a site-specific location in the host Ability to integrate into a site-specific location in the host

chromosome, or to be successfully maintained as stable chromosome, or to be successfully maintained as stable

episomes.episomes.

☻A transcriptional unit that can respond to manipulation of its A transcriptional unit that can respond to manipulation of its

regulatory elementsregulatory elements

☻Ability to target the desired type of cellAbility to target the desired type of cell

☻No components that elicit an immune responseNo components that elicit an immune response

Following 

(1)Interaction of the viral envelope proteins with the host cell surface receptors and loss of the envelope by fusion of the virus membrane with the cell membrane

(2)the virus enters the host cell.

(3)Viral RNA (green) is released into the cytoplasm where it is converted into DNA by reverse transcriptase (blue circle).

(4)The dsDNA enters into the host cell nucleus. However, because retroviral DNA cannot pass the nuclear envelope, it can only integrate into the host genome of cells that are undergoing mitosis.

(5)After nuclear translocation, the viral DNA is integrated into the host genomic DNA by the integrase, and is then termed 'provirus'.

(6) Proviral DNA transcription is performed and mRNA molecules transferred into the cytoplasm,

(7)where they are translated by the cellular machinery.

(8)After assembly of RNA and viral proteins,

(9)progeny viral particles bud from the plasma membrane,

(10)further mature into infectious particles.

rep cap

Replication Capsid

4.8 kb

ITR ITR

Used for immunization against chicken pox.

Therapy is limited to individuals not priorly exposed to this virus.

Affects all kinds of cells.

Transduction efficiency is high.

Large quantity of gene therapy based vaccinations can be developed.

Large pieces of DNA can be incorporated

Based on formation of precipitates when DNA interacts with calcium ions.DNA is mixed with cacl2 and added to phosphate buffer solution. After 20 mins ppt is formed, added to cell in culture. Taken up by endocytosis.

Injects plasmid DNA directly into cell’s nucleus.

Using light microscope, syringe is guided into nucleus and small amount of DNA or RNA is injected.

Injection into cell at specific time of development has potential to alter the genotype and phenotype of cell formed thereafter.

Electroporation is the application of high voltage to a mixture of DNA and cells in suspension.

Duracell

DNA containing the gene of interest

Cell

Protoplast

Power supply

DNA inside the cell

The plant cell with the new gene

Microscopic vesicles in which an aqueous volume is enclosed in a by a membrane composed of lipid molecule.

CATIONIC LIPOSOMES NEGATIVELY CHARGED LIPOSOMES

+ve ly charged particles interact with –ve DNA molecules and form Stable complex.

Have lipids and co-lipids.

Co-lipids are required for stabilization of complexes.

DOPE and DOPC are commonly used co-lipids.

Lipofectin is most commonly used lipid.

Entrap DNA rather than forming complex.

Since both are of same charge, repulsion occurs.

DNA gets entrapped in the aqueous interior.

De-stabilized by low PH.

Highly efficient for in-vivo introduction.

The simplicity of injecting plasmid DNA into muscle with a syringe has greatly influenced many aspects of gene therapy research.

Tissue that show expression are thymus, skin, cardiac, skeletal muscle(long term expression).

Does not undergo replication & does not integrate with host genome upon entry into muscle.

The greatest advantages of intramuscular plasmid DNA injection are its simplicity and the fact that plasmids ranging in size from 2-19 kilo bases have been successfully used to transfer genes to muscle.

DNA coated golden

particlesGene gun

Cell division

Cell with the new gene

cell

Cell’s DNA

Chances of entry into germ cells.

Over expression of gene (Toxicity)

May effect other than intended cells.

Using viral vectors has a fear that once inside the body; the virus will recover its ability to cause its original disease.

For the diagnosis of disease in a fetus/embryo before it is born prenatal testing is performed. If an unborn carries any defective gene then their parent’s definitely want to abort this child this may increase number of abortions.

Genes carried in nanoparticles used to treat cancer in mice.

Cured inherited blindness in humans. Successful in treating adenosine deaminase deficiency

an auto-immune disorder in humans.

GENDICINE: The treatment consists of an adenovirus designed to

insert a gene called p53,codes for a protein that triggers cell suicide when cell start to run amok, preventing them from becoming cancerous. Many tumors arise after the mutation or inactivation of p53, and in cancers of this type restoring the protein shall kill the tumor cells. Going to test on head and neck squamous cancers, as p53 is mutated in 60% of theses tumors.

Who will decide what is normal or disability and what is a disorder??

Who will access to genetic information??

Is somatic therapy more or less ethical than germ cell therapy??

Preliminary attempts in gene therapy proved expensive. Who will have access to these therapies??

Almost every therapy which intended were successful in cell and animal models.

Its mark in treating humans is most awaiting one, as all are in clinical trials.

Lets hope for better way of treating diseases with ethical values.