Gene Therapy:

Treatment which cure disease By introducing DNA into cells

•Major Challenge in G.T Delivery of gene into target cells

Barriers

ExtracellularIntracellular

Why ?

• immune system•Epithelial cells• Degradative Enzymes in blood

• cell membrane• endosomal membrane• degradative enzymes in cytoplasm• Nuclear membrane

Gene Delivery Systems (vehicles): Characteristics =Specific,Efficient,Safe

Viral Gene Carriers Very Efficient

Problems

Non Viral Gene Carriers

Chemical

Physical

•Microinjection•Electroporation•BiolisticsUses chemicals for

delivering gene

• Immunogenic• Random integration of DNA to genome = can result in Cancer• difficult to reproduce viruses for using them as carriers

Basic Mechanism of chemical delivery System:

Polycationic entities + DNA (-vely charge) = Complexes ( +vely charged)

Plasma Membrane (-vely charged)

Electrostatic Interaction

Endosomes

DNA released in ctyosol

DNA Reaches Nucleus

Main Players of Chemical Delivery System:Lipoplexes

PolyplexesLipoplexes: = Complexes of Liposome +DNA

Cationic lipid + Neutral Lipid

Amphiphatic

Hydrophilic head group is attached to hydrophobic domain via a connector

Should be•Chemical stable•Biodegradable

Examples include•Amino acid•Aromatic rings

•Stabilizes the Complexes•Reduces toxicity•The Other Function

Examples include•DOPE•Cholesterol

Structure of Lipoplexes: Self Assembly!!

1. Multilameller Structure: DNA sandwiched, nearly equal head group and Hydrocarbon cross section

2. Hexagonal and inverted Hexagonal: DNA encapsulated in monolayer tubes, large heard group or small head group w.r.t hydrocarbon cross section

Lipoplexes Mediated Transfection Mechanism:

•Rapid mixing of cationic liposome and anionic lipids of endosomes raptures/ destabilizes the endosomes.

•Neutral lipid promotes rupturing

Polyplexes: Complexes of Cationic polymers + DNA

Consist of polymer,DNA binding proteins with ligands attached to them

Examples•Histones•Synthetic polypeptides

Note: Do not have hydrophobic domain •Poly-L-Lysine

•Polyethylinemine

Polyplexes Structure:

•Spherical•Globular•Rod-like•Toroids

Self Assembly!!

Polyplexes Mediated Transfection Mechanism:

•Proton sponge effect that promotes swelling of endosomes which leads to its rupture.

Problems with Chemical Gene Delivery system:

•New Technology so not that efficient yet as compared to viruses.

Summary:

•What is gene therapy•Barriers for Gene Delivery System and Delivery system Characteristics•Viral Gene Delivery System and their problems•Non Viral Gene Delivery System and their Problems•Lipolplexes, Lipoplexes structure and mechanism of Transfection•Polyplexes , Polyplexes structure and mechanism of Transfection

References:

1. Gene delivery by lipoplexes and polyplexes by Conchita Tros de llarduya and et all ( R.paper)

2. Nonviral gene delivery: Techniques and implications for molecular medicines by Alan L.Parker and et all (R.paper)

3. Understanding gene therapy (book) by N.R.Lemoine