Gene Therapy

The invention of recombinant DNA technology consequently led to the immediate inception of engineered gene transfer into human cells, aiming at reversing a cellular dysfunction or creating new cellular function. The concept of direct therapeutic benefit based on a gene defect correction in human cells or on gene therapy was born

Gene therapy relies on Gene intervention Supplementing or replacing defective genes by their normal counterparts At the most basic level: the intracellular delivery of genetic material to generate a therapeutic effect by correcting an existing defect or providing cells with a new function. Initially, only the inherited genetic disorders were in focus but now a wide range of diseases, including cancer, neurodegenerative disorders vascular disease and other acquired diseases are being considered as plausible targets. The ultimate goal of gene therapy is the alleviation of disease upon a single administration of an appropriate therapeutic gene. Hurdle 1. Nucleic acids have poor cell penetration capacity. Vectors are needed Hurdle 2. Humans are equipped with very sophisticated defense systems Integration within the host genome would lead to AE

Germ line vs Somatic gene therapy Germ line therapy vs Somatic therapy Germ-line therapy aims at the introduction of genes into germ-cells or omnipotent embryonal cells (4-8 cellular stage) Considered unethical, nevertheless, lifelong correction Somatic therapy: insertion of genes into diploid cells of an individual where the genetic material is not passed onto its progeny.

Ex vivo vs In vivo gene therapy Ex vivo: delivery is after explanation, cultivation and manipulation in vitro, followed by subsequent reimplantation. ++ minimal complicating immunological problems and enhanced efficiency of vector delivery in vitro. In situ delivery:administering the material directly to the desired tissue is currently the major area of clinical interest )CFTR and cancer gene therapy) In vivo delivery: systemic administration of the delivery systems. Least advanced strategy, rapid clearance mechanisms

Potential targets for Gene Therapy Inherited disorders: an intact version of the gene is introduced into these cells in which inadequate expression of the gene is determining symptom of the disease. Not necessarily 100% correction Stable correction is required: integration is human genome, or episomal with an origin of replication Cystic fibrosis

CANCER TUMOR SUPPRESSOR GENE THERAPY: tumor suppressor gene such as p53 which is mutated in a large number of cancer Gendicine™ is a replication-incompetent adenovirus encoding for the TP53 gene (in place of the viral E1 gene) used for the treatment of a variety of cancers. What makes Gendicine™ interesting is the fact that it became the first gene therapy product that has been approved for clinical use. Glioma, head and neck, pancreatic, and ovarian cancers, demonstrating an acceptable safety profile. Typical complications included fever, injection site pain, nausea, alopecia, leucopenia, and flu-like symptoms

SUICIDE GENE THERAPY: To introduce a transgene encoding for an enzyme that is either absent in mammalian cells or present in a very inactive form, into the tumor cells Herpes Simplex virus-Thymidine kinase. The enzyme phosphorylates the pro-drug ganciclovir By-stander effect!

ANTIANGIOGENIC GENE THERAPY: delivering antiangiogenic factors to the tumour vasculature. Angiogenesis inhibitors that act directly on endothelial cells to cause selective apoptosis of stimulated and proliferating endothelial cells

GENETIC ENHANCEMENT OF ANTITUMOR IMMUNE RESPONSES: Objective is to enhance either the recognition or presentation of tumor-associated antigens. Natural tolerance might be a limitation Genes which encode for artificial receptors, which, when expressed by immune cells, allow these cells to specifically recognize cancer cells thereby increasing the ability of these gene modified immune cells to kill cancer cells in the patient Another way to boost an anti-tumoral immune response: second generation replication-deficient adenovirus of the serotype 5 containing the TNF-α cDNA. Promoter was induced with ionizing radiation

DRUG RESISTANCE GENE THERAPY: Expression of drug-resistance genes in hematopoietic stem cells using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy.

Gene transfer methods The ideal vector for gene delivery would have at least the following characteristics: specificity for the targeted cells; resistance to metabolic degradation and/or attack by the immune system; safety, i.e., minimal side effects; and ability to express, in an appropriately regulated fashion, the therapeutic gene for as long as required.

Gene transfer methods Non viral gene delivery Mechanical Gene Delivery a. Microinjection: the most direct method to introduce gene into cells. Either cytoplasm or nucleus. Microsurgical procedure (needle, precision positioning device, microinjector) visual inspection under microscope. Nuclear vs cytoplasmic injection Very laborious Has been used successfully for skeletal muscles

b. Particle bombardment (gene gun): microparticles coated with genetic material are forcefully injected into cells delivering the functional genes. The particles must be non-toxic, non-reactive, and smaller than the diameter of the target cell (most commonly 1–1.5 um). Naked DNA can be precipitated onto these microparticles, and is then gradually released within the cell post-bombardment. Ex vivo and in vivo could be used

Non viral gene delivery Physical methods Electroporation: very common technique, exposes the cell membrane to high-intensity electrical pulses that can cause transient and localized destabilization of the barrier. Membrane gets highly permeable to exogenous substances. Electric field results in pores. Efficiency is cell dependent

Sonoporation The application of ultrasound. Different frequencies and wave forms. Cavitation causes mechanical perturbation Laser irradiation A laser source with known energy. Focused to the cells via a lens and permeability of the cell is modified by local thermal effect

Kinetics of gene therapy A key advantage of physical methods: direct gene delivery Diffusion of plasmid is slow (size dependent) Internalization is higher than successful transfection Cytoplasmic degradation is possible Electroporation: entry to nucleus is achieved Laser irradiation: nuclear envelope is perforated Ultrasound: acceleration to nucleus Sonoporation: effective nuclear delivery Gene gun: acceleration to nucleus is achieved

Non viral gene delivery Naked DNA (physical and mechanical methods) Polymer based gene delivery Lipid based gene delivery

Polymeric gene delivery Design criteria for synthetic gene delivery Neutralize negatively charged phosphate backbone of DNA to prevent charge repulsion Condense the bulky structure Protect DNA from extracellular and intracellular nucleases

Three packaging strategies Electrostatic interaction: more than one amino group, ionized at neutral pH. Limitation: toxicity, DNA release Encapsulation: within a biodegradable spherical structure. Offers protection. Limitation: formulation factors, low encapsulation efficiency, DNA release Adsorption: adsorption to the surface of biodegradable particles. Surface presentation and enzymes

Cationic lipids and cationic liposomes The most extensively studied DNA carriers Do not encapsulate DNA, form complexes Cationic lipids are amphiphiles, cationic head attached via a linker to hydrocarbon chains Cationic lipids assume various structural phases Could be modified: PEG attachment: stealth, pH sensitive binding Mostly taken up by endocytosis: pH sensitive delivery