14 gene therapy and gene editing

gene therapy

An experimental technique that utilizes genes to treat or prevent diseases by inserting a gene into a patient's cell instead of using medicines/ drugs or surgery.

september 1990

First approved gene therapy experiment occurred on

_______ ____ when Ashanti De Silva was treated

for her heritable disorder called Severe Combined

Immunodeficiency (SCID).

GENE THERAPY

It works by adding new copies of a gene that is broken, or by replacing a defective or missing gene in a patient's cells with a healthy version of that gene.

Both inherited genetic diseases (e.g., hemophilia and sickle cell disease) and acquired disorders (e.g., leukemia) have been treated

EX VIVO GENE THERAPY

isolate cells with genetic defect from a patient

grow the cells in culture

introduce the therapeutic genes

select genetically corrected cells and grow

transplant the modified cells to the patient

IN VIVO GENE THERAPY

Direct delivery of therapeutic gene into a target cell into patients body.

Carried out by viral or non viral vector systems.

It can be the only possible options in patients where

individual cells cannot be cultured in vitro in sufficient

numbers (e.g brain cells).

necessary when cultured cells cannot be re-implanted in patients effectively.

germ line

somatic

types of gene therapy

germ line gene therapy

Therapeutic genes are transferred into the germ cells. E.g. Introduction of genes into eggs and sperms

It is inheritable and can be passed on to later generations.

For safety, ethical and technical reasons, it is not being attempted at present.

Somatic Gene Therapy

Therapeutic genes are transferred into the defective cells.

It does not prevent the disease from occurring in the next generation.

It has to be done several times over the coarse of the patients life because the effects do not last very long.

viral and non viral

vectors in gene therapy

viral vectors

adeno

retro

adeno associated

adeno virus

good vector

Target: non dividing human cell

Common cold adenovirus

retro virus

The recombinant retroviruses have the ability to integrate into the host genome in a stable fashion.

Can carry a DNA of size of less than 3.4kb

Replication defective virus particles

Target: dividing cell

Adeno Associated Virus

Human virus that can integrate into chromosome 19

Single stranded, non pathogenic small DNA virus

AAV enters host cell, becomes double stranded and gets integrated into chromosome.

non viral vectors

pure DNA construct

lipoplexes

DNA molecular conjugates

human artificial chromosome

Pure DNA Construct

Direct introduction of pure DNA construct into target tissue.

Lipoplexes

Lipid DNA complexes; DNA construct surrounded by artificial lipid layer.

Most of it gets degraded by lysosomes.

DNA Molecular Conjugates

Commonly used synthetic conjugates is poly-L-lysine bound to specific target cell receptor.

Therapeutic DNA is then made to combine with the conjugate to form a complex.

It avoids lysosomal breakdown of DNA.

Human Artificial Chromosome

Can carry a large DNA, with one or more therapeutic genes with regulatory elements.

gene gun

microinjection

methods of gene delivery (physical)

Gene Gun

employs a high-pressure delivery system to shoot tissue with gold or tungsten particles that are coated with DNA.

Microinjection

process of using a glass micropipette to insert microscopic substances into a single living cell.

detergent mixtures

lipofection

methods of gene delivery (chemical)

Using detergent mixtures

Certain charged chemical compounds like Calcium phosphates are mixed with functional cDNA of desired function.

The mixture is introduced near the vicinity of recipient cells.

The chemical disturbs the cell membrane, widens the pore size and allows cDNA to pass through the cell.

Lipofection

It is a technique used to inject genetic materials into a cell by means of liposomes.

Liposomes are artificial phospholipid vesicles used to deliver a variety of molecules including DNA into the cells.

Gene Augmentation Therapy

Gene Inhibition Therapy

other types of gene therapy

Gene Augmentation Therapy

• Most commonly form of gene therapy.

• Foreign gene replaces missing or defective gene.

Replacement of defective p53 gene by a normal one in liver cancer

Gene Inhibition Therapy

• Done to block the overproduction of some proteins.

antigene

antisense

2 types of Gene Inhibition Therapy

Antigene

blocks transcription using antigene oligonucleotide

Antisense

blocks translation using antisense oligonucleotide

GENE EDITING

Strategies and techniques developed for the targeted, specific modification of the genetic information or genome of living organisms.

Disruption, insertion, replacement at locus in the genome

Insertion

to obtain a new function

to compensate for a defective gene

Inactivation or "knock-out"

Fundamental research to know function of a gene

To remove a persistent viral sequence from infected cells

In agriculture to eliminate the irritant or allergenic properties of a plant

Correction

To remove and replace a defective gene sequence with a functional sequence

Improve the properties of a species without the addition of foreign DNA.