Gene therapy

1
Gene therapy

• Class presentation
• Nanobiotechnology 2004

2
Gene therapy

• Use of genes to treat disease
• Several approaches
• The fundamental idea is to administer a
  functional gene, so as to give targeted cells a
  new protein-manufacturing capacity, one that
  would have been present but is missing or
  defective, or sometimes never meant to be there.
• For correcting faulty genes
• A normal gene may be inserted into a nonspecific
  location within the genome to replace a
  nonfunctional gene (most common).
• The abnormal gene could be repaired through
  selective reverse mutation, which returns the
  gene to its normal function.
• The regulation of a particular gene could be
  altered.
• Some diseases can also be treated by interfering
  with the activity of the sick gene
• Cells normally switch genes on and off by
  attaching certain chemicals to the gene. One
  therapeutic approach might be to exploit this
  process with a drug that switches a harmful gene
  off.
• Insert some new DNA into the middle of the
  harmful gene.
• Insert a gene that is the exact chemical opposite
  of the sick gene. The product of this
  "anti-sense" gene would neutralize the product of
  the sick gene.

3
Why is gene therapy hard?

• The gene
• has to be designed
• needs to be manufactured
• has to be directed to the right cells in the
  body.
• needs to be controlled, so the protein is
  produced only when it is needed.
• Each of these problems has one or more
  solutions.
• Together, however, they have so far prevented
  success in all but a few single-gene diseases.

4
Vectors

• Viral vectors
• DNA virus
• RNA virus
• Non-viral vectors
• liposomes
• polimers
• nanodiveces

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• RNA vectors
• A class of viruses that can create
  double-stranded DNA copies of their RNA genomes.
  These copies of its genome can be integrated into
  the genome of host cells. Human immunodeficiency
  virus (HIV) is a retrovirus.
• efficient gene transfer into many cell types and
  can stably integrate into the host cell genome
• have minimal risk because retroviruses have
  evolved into relatively non-pathogenic parasites
  (expection e.g. HIV)
• accept up to about 8 kb of exogenous DNA.
• are not inactivated by human serum, and
  transduce dividing cells.

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• DNA vectors
• Adenovirus
• The DNA virus used most widely for in situ gene
  transfer vectors
• inserts up to 35 kb
• they are human viruses and are able to transduce
  a large number of different human cell types at
  very high efficiency
• can transduce non-dividing cells.
• AAV
• non-pathogenic virus that is widespread in the
  human population
• Unfortunately appear to integrate in a
  nonspecific manner
• room for about 4.8 kb of added DNA
• Other types
• HEPES
• hybrid systems have been reported where an
  adenoviral vector is used to carry a retroviral
  vector into a cell that is normally inaccessible
  to retroviral transduction

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Non viral vectors

• two factors suggest that non-viral gene delivery
  systems will be the preferred choice in the
  future
• ease of manufacturing
• more flexible with regard to size of the DNA
  being trasferred
• generally safer in vivo
• do not elicit a specific immno response and can
  therefor be administered repeatedly
• but less efficient en delivering DNA particularly
  when used in vivo
• major type
• cationic phosfolipids
• cationic polymers

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• Liposomes
• low immunogenicity but toxicity of lipids and low
  transfection efficiency.
• Polimer-based systems
• e.g. Using collagen, lactic or glycolic acid,
  polyanhdride or polyethylene vinyl coacetate
• DNA encapsulation within the polymer can protect
  againgst degradation until release
• release form the polymer and into the tissue can
  be designed to occur rapidly or over extended
  period of time thus the delivery system can be
  tailored to a particular application.
• Intrinsic drawbacks with cationic carriers such
  as solubility, cytotoxicity and low trasfection
  efficiendy, have limietd their use in vivo. These
  vectors sometimes attract serum proteins
  resulting in dynamic changes in their
  physicochemical properties.

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• Nano devices
• novel delivery sistems which can be administere
  in novel ways (e.g. Aerosols) are being
  developed. The smaller the size of the condensed
  DNA particles, the better the in vivo diffusion
  towards target cells and the trafficing within
  the cell.
• chemical biological hybrid composed of
  oligonucleotide DNA, RNA or peptide covalently
  attached to nanoparticle (TiO2 4.3 nm).
• Could attach one molecule of DNA or pepetide that
  could target it ot a particular cellular site,
  and another peptide that can carry out an
  effective function.
• Ni-Au devices

10
What factors have kept gene therapy from becoming
an effective treatment for genetic disease?

• Short-lived nature of gene therapy - Before gene
  therapy can become a permanent cure for any
  condition, the therapeutic DNA introduced into
  target cells must remain functional and the cells
  containing the therapeutic DNA must be long-lived
  and stable. Problems with integrating therapeutic
  DNA into the genome and the rapidly dividing
  nature of many cells prevent gene therapy from
  achieving any long-term benefits.
• Problems with viral vectors - Viruses, present a
  variety of potential problems to the patient
  --toxicity, immune and inflammatory responses,
  and gene control and targeting issues. There is
  always the fear that the viral vector, once
  inside the patient, may recover its ability to
  cause disease.
• Multigene disorders - Conditions or disorders
  that arise from mutations in a single gene are
  the best candidates for gene therapy.
  Unfortunately, some the most commonly occurring
  disorders, such as heart disease, high blood
  pressure, Alzheimer's disease, arthritis, and
  diabetes, are caused by the combined effects of
  variations in many genes.

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Gene therapy and cancer

• Researchers are studying several ways to treat
  cancer using gene therapy. Some approaches target
  healthy cells to enhance their ability to fight
  cancer. Other approaches target cancer cells, to
  destroy them or prevent their growth. Some gene
  therapy techniques under study are described
  below.
• replace missing or altered genes with healthy
  genes.
• stimulate the bodys natural ability to attack
  cancer cells.
• In some studies, scientists inject cancer cells
  with genes that make them more sensitive
  treatments.
• suicide genes are introduced into cancer cells.
  Later, a pro-drug (an inactive form of a drug)
  is given to the patient. The pro-drug is
  activated in cancer cells containing these
  suicide genes, which leads to the destruction
  of those cancer cells.
• Other research is focused on the use of gene
  therapy to prevent cancer cells from developing
  new blood vessels.

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Clinical trials

• Phase I trialare the first step in testing a new
  approach in humans. In these studies, researchers
  evaluate what dose is safe, how a new agent
  should be given (by mouth, injected into a vein,
  or injected into the muscle), and how often.
  Researchers watch closely for any harmful side
  effects. Phase I trials usually enroll a small
  number of patients and take place at only a few
  locations.
• Phase II trial study the safety and effectiveness
  of an agent or intervention, and evaluate how it
  affects the human body. Phase II studies usually
  focus on a particular type of cancer, and include
  fewer than 100 patients.
• Phase III trial compare a new agent or
  intervention (or new use of a standard one) with
  the currentstandard therapy. In most cases,
  studies move into phase III testing only after
  they have shown promise in phases I and II. Phase
  III trials may include hundreds of people across
  the country.
• Phase IV trial are conducted to further evaluate
  the long-term safety and effectiveness of a
  treatment. They usually take place after the
  treatment has been approved for standard use.
  Several hundred to several thousand people may
  take part in a phase IV study. These studies are
  less common than phase I, II, or III trials.

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Clinical trials

• At present over 300 clinical protocols have been
  approved
• Only one phase III and most of the rest of the
  approved gene therapy protocols are for smaller
  phase I/II trials.
• Genetic Therapy Inc./Novartis is carrying out
  the phase-III clinical trial. The target disease
  is glioblastoma multiforma, a malignant brain
  tumour. The rationale is to insert a gene capable
  of directing cell killing into the tumour while
  protecting the normal brain cells.
• The retroviral vector used (G1TkSvNa) contains
  the neomycin-resistance gene as a selective
  marker and the herpes simplex thymidine kinase
  (HSTK) gene. The actual material injected into
  the tumour mass is a mouse producer cell line
  (PA317) which generates retroviral particles
  carrying the G1TkSvNa vector. As the only
  dividing cells in the area of a growing brain
  tumour are the tumour cells and cells of the
  vasculature supplying blood to the tumour, and
  retroviral vectors only transduce dividing cells,
  the only cells to receive the vector should be
  the cells of the tumour and its blood vessels.
• In theory, the tumour cells that have been
  transduced with the vector containing the HSTK
  blocks the DNA synthesis machinery and kills the
  cells. In fact, at least four distinct mechanisms
  contribute to tumour cell death in this protocol.
  
• Several phase II trials are underway testing
  gene-therapy vectors as 'vaccines', either
  against cancer (48) or against AIDS (49).

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W. French Anderson, who led the first gene
therapy trial in 1990, thinks gene therapy will
outgrow its problems just like other technologies
have. In the journal Science, he wrote The
field of gene therapy has been criticized for
promising too much and providing too little
during its first 10 years of existence. But gene
therapy, like every other major new technology,
takes time to develop. Antibiotics, monoclonal
antibodies, organ transplants, to name just a few
areas of medicine, have taken many years to
mature. Early hopes are always frustrated by the
many incremental steps necessary to produce
"success." Gene therapy will succeed with time.
And it is important that it does succeed, because
no other area of medicine holds as much promise
for providing cures for the many devastating
diseases that now ravage humankind.