1. Gene Delivery Ali Badiee (Pharm. D., Ph.D.) School of Pharmacy,
Mashhad University of Medical Sciences,
Iran

2. Gene Therapy
A method to introduce genetic materials into cells for the production of therapeutic proteins or blocking the synthesis of harmful proteins.
For the correction of genetic defects in target cells. The treatment of diseases with single gene disorders (e.g., cystic fibrosis, haemophilia, sickle cell anaemia, M-thalassemia) and malignant tumours.
For the destruction of target cells using a cytotoxic pathway. The treatment of malignant tumours and DNA vaccination.

3. Gene Therapy
ex vivo GT
in vivo GT

5. Approaches for cancer GT
Introduction of cytokine genes
Introduction of Suicide genes
Introduction of tumor suppressor genes
Inserting a gene that confers drug resistance in hematopoietic stem cells to protect them

7. Research fields in gene therapy
Development of a therapeutic gene
Development of naked pDNA delivery methods
Development of a safe and efficient gene delivery system

8. In vivo fate of pDNA

9. Naked pDNA delivery methods
Mechanical
Microinjection
Particle bombardment (gene gun)
Non-mechanical
Electroporation

10. Mechanical strategies for pDNA into cells

11. Gene gun delivery
helium gas
Dose μg

12. Gene delivery systems Viral vectors Non-viral vectors Retroviruses
Adenoviruses
etc.
Non-viral vectors
Lipid-based (lipoplex)
Liposomal
Non-liposomal
Polymer-based (ployplex)
Polyethylenimines (PEI)
Cationic block copolymers
Polyethylene glycols (PEG)
Cyclodextrins
Dendritic polyamidoamines

14. Viral vectors
Are the most effective means of DNA delivery, with high efficiencies (usually >90%) for both delivery and expression.
around 75% of recent clinical protocols involving gene therapy use recombinant virus-based vectors for DNA delivery
Drawbacks with viral vectors:
High risk of mutagenicity
Immunogenicity
Low production yield
Limited gene payload size
Oncogene activation
Production and packaging problems
High cost, etc.

15. Non-viral vectors Advantages: Disadvantage: Low cytotoxicity
Low immunogenicity
No size limit
Low cost
Reproducibility
Disadvantage:
Low transfection efficacy

17. For drug formulators:
DNA (pro-drug) is a 5,000 bp plasmid that has a molecular weight of about 3,300,000 Daltons and carries 10,000 negative charges.
DNA has 3 major problems:
Low uptake across the plasma membrane
Limited stability in cytoplasm
Lack of nuclear targeting

18. Gene delivery systems At least operate at one of three general levels:
DNA condensation and complexation
Endocytosis
Nuclear targeting/entry

19. Gene delivery systems Viral vectors Non-viral vectors Retroviruses
Adenoviruses
etc.
Non-viral vectors
Lipid-based (lipoplex)
Liposomal
Non-liposomal
Polymer-based (ployplex)
Polyethylenimines (PEI)
Cationic block copolymers
Polyethylene glycols (PEG)
Cyclodextrins
Dendritic polyamidoamines

20. Lipoplex
Preparation: Simple mixing of preformed cationic liposomes and DNA in an aqueous solution.
Electrostatic interactions between the positive charges of the cationic lipid head groups and the phosphate DNA backbones are the main driving force for the lipoplex formation
Concentration, temperature, environment, and kinetics of mixing should be considered carefully in any protocol
Preparation with a slight excess of positive charges confers a higher transfection efficiency (molar ratio L/D: ~1.2)
Size: nm ( High charge (P or N) => reduce size, more homogeneous)

21. Liposomal Non-liposomal Usually contain two types of lipids
a cationic lipid (a positively charged amphiphile) for DNA condensation and cellular membrane interaction (DOTMA)
a neutral helper lipid, to increase transfection efficiency as it has a membrane fusion promoting ability (DOPE)
Non-liposomal
Lipopolyamines combine both the characteristics of cationic and helper lipids (DOGS (polyamine spermine)).

22. The first reported cationic lipid was
DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride)
Cationic lipids consist of:
a hydrophilic headgroup which is positively charged, usually via the protonation of one (monovalent lipid) or several (multivalent lipid) amino groups
a hydrophobic portion composed of a steroid or of alkyl chains (saturated or unsaturated)
a linker whose nature and length may impact on the stability and the biodegradability of the vector (ether > carbamate > ester)

24. Polyplex The complexe of polycations and DNA
To be able to present different:
lengths
geometry (linear versus branched)
Substitutions
additions of functional groups
Extensive structure/function relationship studies

25. Different types of polyplexes
Natural:
Proteins (Histones, Protamine)
Carbohydrate-based (Chitosan, Cyclodextrin)
Synthetic:
PEI
Amino acid polymers (Polylysine, Polyornithine)
Cationic dendrimers (polyamidoamine or PAMAM)
Block co-polymers (poloxamer)

28. Formulation
It is performed at ionic strength where the polycation/polyanion association is rapid and almost irreversible.
The sizes of polyplex increase when
charge ratio goes toward 1
ionic strength of the formulation buffer increases
Increasing charge ratio (N/P) to >1 => compaction of DNA improves
At a charge ratio of 1 (N/P):
the DNA is fully bound, compacted, aggregate and show low solubility

29. Formulation
Highly charged particles rapidly aggregate in high ionic strength solutions (blood) owing to
the decrease in the protective electrostatic double layer
Strategies to increase the solubility or to reduce polyplex aggregation
development of copolymers bearing hydrophilic segments
hydrophilic shell at the exterior of the polyplexes
prevents aggregation by sterical repulsion
enhances the aqueous solubility

30. PEI PEI is a nonbiodegradable & cytotoxic polymer
Are one of the highest transfection efficiencies
densely charged polymers: one third of the atoms are nitrogen, and one sixth of the nitrogen atoms carry a positive charge at physiological pH.
For complete complexation of DNA, a (N/P) charge ratio of at least 2 is required.
For transfection, N/P ratios of >5 are usually applied
The “proton sponge” character of PEI
Protonation of amine groups depends on the pH
Buffering effect by the amine groups on PEI keeps the endosomal pH neutral, which results in osmotic bursting of the endosomes and release of the polyplexes in the cytosol of the cells.
PEI is a nonbiodegradable & cytotoxic polymer

31. Comparison of cationic lipids with polymers:
No hydrophobic moiety
Completely soluble in water
More efficient to condense DNA
More toxic

32. Next generation of cationic polymers
Current developments of polycationic carriers have two major aims:
to generate backbones that mediate higher transfection efficiency than existing carriers
to make carriers less toxic, more biocompatible, and biodegradable
Combining lipids and polymers to form new vectors (i.e., lipopolyplexe)
LPD

33. LPD

34. in vivo fate of NVGDS OR Barriers for NVGDS
From injection site to the surface of target cell (Extracellular barriers)
From the surface of target cell to the nucleus (Intracellular barriers)

35. Extracellular barriers
Lipoplexes and polyplexes are colloidal suspensions of DNA that must be stabilized to remain discrete particles in the blood to get ride of hepatic & lung clearance
They must show
Low toxicity (low M.W. polymer => low toxicity)
Minimize interactions with plasma proteins, extracellular matrices and non-targeted cell surfaces (non-self interactions) (not strong positive charge or PEGylation)
Evade the adaptive immune system (PEGylation or reduce the size)
not aggregate (self interactions) (PEGylation)

36. Intracellular barriers

37. Possible mechanisms for endosomal escape
1. Disruption of endosomal membrane through mixing with the cationic lipid vector
2. Cationic lipid interaction with the negatively charged endosomal membrane.
3. Proton sponge hypothesis, where a cationic lipid, has the ability of endosomal pH buffering that leads to chloride ion influx together with water molecules (H3O+) that leads to membrane swelling and disruption.
4. Membrane destabilising peptides, which includes fusogenic or lytic peptides, such as the basic peptide KALA, acidic peptide EGLA, or the uncharged peptides at neutral pH such as H5WYG . These peptides have the ability to disrupt the endosomal membrane and increase the release of DNA to the cytosol.

38. Intracellular barriers