Download S05 Biotechnology Gene Therapy 1

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
SUICIDE GENE THERAPY: Herpes Simplex virusThymidine kinase. The enzyme phosphorylates the
pro-drug ganciclovir
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: 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
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 nontoxic, 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 highintensity 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
1. Naked DNA (physical and mechanical
methods)
2. Polymer based gene delivery
3. Lipid based gene delivery
Polymeric gene delivery
• Design criteria for synthetic gene delivery
1. Neutralize negatively charged phosphate
backbone of DNA to prevent charge
repulsion
2. Condense the bulky structure
3. Protect DNA from extracellular and
intracellular nucleases
• Three packaging strategies
1. Electrostatic interaction: more than one amino
group, ionized at neutral pH. Limitation:
toxicity, DNA release
2. Encapsulation: within a biodegradable
spherical structure. Offers protection.
Limitation: formulation factors, low
encapsulation efficiency, DNA release
3. 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