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PhD Course . TOPICS IN (NANO) BIOTECHNOLOGY Gene Therapy Lecture 11. 27th November, 2006. What is gene therapy? Why is it used?. Gene therapy is the application of genetic principles in the treatment of human disease

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PhD Course


Gene Therapy

Lecture 11

27th November, 2006


What is gene therapy? Why is it used?

  • Gene therapy is the application of genetic principles in the treatment of human disease
  • Gene therapy = Introduction of genetic material into normal cells in order to:
    • counteract the effect of a disease gene or
    • introduce a new function
  • GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized  to improve a genetic disorder
  • Can be applied as therapy for cancers, inherited disorders, infectious diseases, immune system disorders

History of gene therapy

1930’s “genetic engineering” - plant/animal breeding

60’s first ideas of using genes therapeutically

50’s-70’s gene transfer developed

70’s-80’s recombinant DNA technology

1990 first GT in humans (ADA deficiency)

2001 596 GT clinical trials (3464 patients)


Three types of gene therapy:

  • Monogenic gene therapy
    • Provides genes to encode for the production of a specific protein
      • Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, SCID
  • Suicide gene therapy
    • Provide ‘suicide’ genes to target cancer cells for destruction
      • Cancer
  • Antisense gene therapy
    • Provides a single stranded gene in an’antisense’ (backward) orientation to block the production of harmful proteins
      • AIDS/HIV
different delivery systems are available
Different Delivery Systems are Available
  • In vivo versus ex vivo
    • In vivo = delivery of genes takes place in the body
    • Ex vivo = delivery takes place out of the body, and then cells are placed back into the body

Getting genes into cells

  • In vivo versus ex vivo
    • In vivo = intravenous or intramuscular or non-invasive (‘sniffable’)
    • Ex vivo = hepatocytes, skin fibroblasts, haematopoietic cells (‘bioreactors’)
  • Gene delivery approaches
    • Physical methods
    • Non-viral vectors
    • Viral vectors

In vivo techniques

  • In vivo techniques usually utilize viral vectors
    • Virus = carrier of desired gene
    • Virus is usually “crippled” to disable its ability to cause disease
    • Viral methods have proved to be the most efficient to date
    • Many viral vectors can stable integrate the desired gene into the target cell’s genome
  • Problem: Replication defective viruses adversely affect the virus’ normal ability to spread genes in the body
    • Reliant on diffusion and spread
    • Hampered by small intercellular spaces for transport
    • Restricted by viral-binding ligands on cell surface  therefore cannot advance far.

Viral vectors

“Viruses are highly evolved

natural vectors

for the transfer of foreign genetic information

into cells”

Kay et al 2001

But to improve safety,

they need to be replication defective


Viral vectors

Compared to naked

DNA, virus particles

provide a relatively

efficient means of

transporting DNA into cells, for expression in the nucleus as recombinant genes

(example = adenovirus).

[figure from Flint et al. Principles of Virology, ASM Press, 2000]


Viral vectors

  • Retroviruses
    • eg Moloney murine leukaemia virus


    • Lentiviruses (eg HIV, SIV)
  • Adenoviruses
  • Herpes simplex
  • Adeno-associated viruses (AAV)

Engineering a virus into a viral vector

Vector DNA

Helper DNA

essential viralgenes



Viral vector




wildtype virus

Packaging cell




Gene transfer

Vector uncoating



Integrated expression


Episomal vector

Therapeutic mRNA

and protein

Target cell


Delivery System of Choice = Viral Vectors

A. Rendering virus vector harmless

  • Remove harmful genes  “cripple” the virus
  • Example – removal of env gene  virus is not capable of producing a functional envelope
  • Vectors needed in very large numbers to achieve successful delivery of new genes into patient’s cells
    • Vectors must be propagated in large numbers in cell culture (109) with the aid of a helper virus

Delivery System of Choice = Viral Vectors

B. Integrating versus Non-Integrating Viruses

  • Integrating viruses
    • Retrovirus (e.g. murine leukemia virus)
    • Adeno-associated virus (only 4kbp accommodated)
    • Lentivirus
  • Non-Integrating viruses
    • Adenovirus
    • Alphavirus
    • Herpes Simplex Virus
    • Vaccinia


  • Advantages
  • High transduction efficiency
  • Insert size up to 8kbHigh viral titer (1010-1013)
  • Infects both replicating and differentiated cells
  • Disadvantages
  • Expression is transient (viral DNA does not integrate)
  • Viral proteins can be expressed in host following vector administration
  • In vivo delivery hampered by host immune response

Herpes Simplex Virus

  • Advantages
  • Large insert size
  • Could provide long- term CNS gene expression
  • High titer
  • Disadvantages
  • System currently under development
  • Current vectors provide transient expression
  • Low transduction efficiency

Ex vivo

  • Ex vivo manipulation techniques
    • Electroporation
    • Liposomes
    • Calcium phosphate
    • Gold bullets (fired within helium pressurized gun)
    • Retrotransposons (jumping genes – early days)
    • Human artificial chromosomes

A phospholipid


Lipid Organization

Phopholipid Hierarchal Structures

  • In aqueous solution, polar phospholipids form ordered aggregates to minimize hydrophobic interactions
  • Lipid shape and conditions of formation affect the final lipid organized structure


  • Liposomes are
    • not limited by size or number of genes
    • safe
    • easy to produce
    • short-term expression







  • Diverse manners of ‘lysing’ the liposome
    • Temperature sensitive
    • Target sensitive
    • pH sensitive
    • Electric field sensitive
limitations of gene therapy
Limitations of Gene Therapy
  • Gene delivery
    • Limited tropism of viral vectors
    • Dependence on cell cycle by some viral vectors (i.e. mitosis required)
  • Duration of gene activity
    • Non-integrating delivery will be transient (transient expression)
    • Integrated delivery will be stable
  • Patient safety
    • Immune hyperresponsiveness (hypersensitivity reactions directed against viral vector components or against transgenes expressed in treated cells)
    • Integration is not controlled  oncogenes may be involved at insertion point  cancer?

Limitations of Gene Therapy

  • Gene control/regulation
    • Most viral vectors are unable to accommodate full length human genes containing all of their original regulatory sequences
    • Human cDNA often used  much regulatory information is lost (e.g. enhancers inside introns)
    • Often promoters are substituted  therefore gene expression pattern may be very different
    • Random integration can adversely affect expression (insertion near highly methylated heterogeneous DNA may silence gene expression)

Limitations of Gene Therapy

Gene Therapy Trials in U.S.

(Information from US NIH, Office of

Recombinant DNA Activities – 1999)


# Trials (total = 338)

Genetic disease








  • Expense
    • Costly because of cell culturing needs involved in ex vivo techniques
    • Virus cultures for in vivo delivery
    • Usually the number of patients enrolled in any given trial is <20
    • More than 5000 patients have been treated in last ~12 years worldwide
example severe combined immunodeficiency disease scid
Example: Severe Combined Immunodeficiency Disease (SCID)
  • Before GT, patients received a bone marrow transplant
    • David, the “Boy in the Bubble”, received BM from his sister  unfortunately he died from a a form of blood cancer

Example: Severe Combined Immunodeficiency Disease (SCID)

  • SCID is caused by an Adenosine Deaminase Deficiency (ADA)
    • Gene is located on chromosome #22 (32 Kbp, 12 exons)
    • Deficiency results in failure to develop functional T and B lymphocytes
    • ADA is involved in purine degradation
    • Accumulation of nucleotide metabolites = TOXIC to developing T lymphocytes
    • B cells don’t mature because they require T cell help
    • Patients cannot withstand infection  die if untreated
example severe combined immunodeficiency disease scid1
Example: Severe Combined Immunodeficiency Disease (SCID)
  • September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial
    • Ashanti (4 year old girl)
      • Her lymphocytes were gene-altered (~109) ex vivo used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used)
    • Cynthia (9 year old girl) treated in same year
  • Problem: WBC are short-lived, therefore treatment must be repeated regularly
ornithine transcarbamylase otc deficiency
Ornithine transcarbamylase (OTC) deficiency
  • September 17, 1999
    • Ornithine transcarbamylase (OTC) deficiency
      • Urea cycle disorder (1/10,000 births)
      • Encoded on X chromosome
        • Females usually carriers, sons have disease
    • Urea cycle = series of 5 liver enzymes that rid the body of ammonia (toxic breakdown product of protein)
      • If enzymes are missing or deficient, ammonia accumulates in the blood and travels to the brain (coma, brain damage or death)
ornithine transcarbamylase otc deficiency1
Ornithine transcarbamylase (OTC) deficiency
  • Severe OTC deficiency
    • Newborns  coma within 72 hours
      • Most suffer severe brain damage
      • ½ die in first month
      • ½ of survivors die by age 5
    • Early treatment
      • Low-protein formula called “keto-acid”
    • Modern day treatment
      • Sodium benzoate and another sodium derivative
      • Bind ammonia  helps eliminate it from the body
ornithine transcarbamylase otc deficiency2
Ornithine transcarbamylase (OTC) deficiency
  • Case study: Jesse Gelsinger
    • GT began Sept. 13, 1999, Coma on Sept. 14, Brain dead and life support terminated on Sept. 17, 1999
    • Cause of death: Respiratory Disease Syndrome
    • Adenovirus (a weakened cold virus) was the vector of choice (DNA genome and an icosahedral capsid)
    • Chain reaction occurred that previous testing had not predicted following introduction of “maximum tolerated dose”
      • Jaundice, kidney failure, lung failure and brain death
      • Adenovirus triggered an overwhelming inflammatory reaction  massive production of monokine IL-6

 multiple organ failure

single gene defects most attractive candidates
Single Gene Defects = Most Attractive Candidates
  • Cystic fibrosis
    • “Crippled” adenovirus selected (non-integrating, replication defective, respiratory virus)
    • Gene therapy trials – 3 Research teams, 10 patients/team
      • 2 teams administered virus via aerosol delivery into nasal passages ad lungs
      • 1 team administered virus via nasal passages only
      • Only transient expression observed  because adenovirus does not integrate into genome like retroviruses
single gene defects most attractive candidates1
Single Gene Defects = Most Attractive Candidates
  • AIDS
    • HIV patients  T lymphocytes treated ex vivo with rev and env defective mutant strains of HIV
    • Large numbers of cells obtained
      • Injected back into patient
      • Stimulated good CD8+ cytotoxic T cell responses (Tcyt)
single gene defects most attractive candidates2
Single Gene Defects = Most Attractive Candidates
  • Familial Hypercholesterolemia
    • Defective cholesterol receptors on liver cells
      • Fail to filter cholesterol from blood properly
      • Cholesterol levels are elevated, increasing risk of heart attacks and strokes
    • 1993  First attempt
      • Retroviral vector used to infect 3.2 x 109 liver cells (~15% of patients liver) ex vivo
        • Infused back into patient
        • Improvement seen
    • Has been used in many trials since then


  • A sample of bone marrow is removed.
  • Stem cells are isolated and allowed to multiply in culture.
  • Cells are treated with a modified virus containing a therapeutic gene


  • The virus is taken up by individual cells and the therapeutic gene goes into the cell's nucleus.
  • Treated ("corrected") cells are injected into the bloodstream.
  • Treated cells respond to injury signals from degenerating muscle or other tissues and migrate out of the bloodstream.
  • Treated cells patch damage and build healthy tissue

Recent Developments in Gene Therapy

  • Liposomes coated in polymer PEG – can cross the blood-brain barrier (viral vectors are too big) (January 2003)
  • Case Western Uni. & Copernicus Therapeutics able to create tiny liposomes 25nm across to carry therapeutic DNA through pores in nuclear membrane
  • New gene approach repairs errors in mRNA
    • Thalassaemia
    • Cystic fibrosis
    • Some cancers
    • (Please refer to


  • 2003 – temporary hold on all gene therapy trials including retroviral vectors in blood stem cells
  • Too early to tell
  • $200 million/year by NIH on clinical trials
  • Desperately need improved DELIVERY …could liposomes be the answer?