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

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.

Gene Therapy ex vivo GT in vivo GT

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

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

In vivo fate of pDNA

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

Mechanical strategies for pDNA into cells

Gene gun delivery helium gas Dose 0.1- 1 μg

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

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.

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

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

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

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

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: 100-200 nm ( High charge (P or N) => reduce size, more homogeneous)

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)).

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)

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

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)

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

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

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

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

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

LPD

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)

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)

Intracellular barriers

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.

Intracellular barriers