Biology 311 Human Genetics
Fall 2004
Lecture: Gene Therapy
Readings:
Mountain, A. (2000) Gene therapy: the first decade. TIBTECH 18, 119-128.
Wu, N. and Ataai, M. M. (2000) Production of viral vectors for gene therapy
applications. Current opinion in biotechnology 11, 205-208.
Chapter 21 pp. 616-629
Presentation:
Presentation on gene therapy by Nicholas Simon Spring 2004, Biology of Cancer
Outline:
1. Gene therapy strategies
2. Disease targets
3. Gene therapy vectors
a. viruses
b. non-viral
4. Future prospects
Lecture:
1. Gene therapy strategies
Gene therapy= The treatment or prevention of disease by gene transfer.
Two major approaches:
a. Gene addition (supplementation): Introduction of a new copy of a gene to
supplement an existing copy. Gene therapy protocols in clinical trials all use this
strategy.
b. Gene replacement: Correction or replacement of a defective gene by a
functioning gene. Technically much more difficult.
Other strategies are shown in Fig. 21.4
2. Disease targets
 Existing therapies are directed at somatic cells, not germ line (gamete)
cells.
 Cancer cells are a major target; most gene therapies for cancer introduce
versions of the tumor suppressor gene p53 in order to restore normal cell
control.
 Some diseases caused by single gene defects (monogenic diseases) are
targets for gene therapy. For example, cystic fibrosis.
 Some infectious diseases are being treated with gene therapy
approaches.
 Some genetic diseases are not readily approachable with gene therapies
yet due to the large size of the gene (muscular dystrophy) or inability of
the vectors to direct the gene to the correct location.
Drug and therapeutic testing: Clinical trials
Phase I: Small trials of 10-15 individuals to determine toxicity of a treatment.
Phase II: Small trials of 10-50 individuals to determine toxicity, to test possible
dosages or delivery methods, and to obtain preliminary results on effectiveness.
Phase III: Large trials of several hundred to a thousand individuals to determine
whether a treatment strategy is safe and effective. A successful trial provides
results that lead to approval of drugs or more widespread applications of new
therapies.
3. Gene therapy vectors
a. viruses
Adenovirus:
 Human virus, causes respiratory tract illness
 Double stranded DNA virus of 36 kb
 Can be grown to high titer
 Can infect a variety of cell types
 Infection can trigger an adverse immune response
 Size limit of introduced gene about 7.5 kb
 Most successful vector so far for human gene therapies
 Most useful versions are gutted, removing viral genes that trigger the
immune response, but require growth in presence of helper virus
 Short duration of expression
Adeno-associated virus (AAV):
 Human virus, not toxic to infected cells, doesn't trigger an immune
response
 A single-stranded DNA virus with genome 4680 bases long
 Replication requires functions of a helper virus such as adenovirus or
herpes simplex
 Can infect both dividing and non-dividing cells
 Can only carry about a 4.5 kb gene
 Low production yield
 New generation vectors engineer helper functions on other plasmids or
viruses
Retroviral vectors:
 Most are based on a mouse leukemia virus (MMLV=Moloney murine
leukemia virus)
 Only infect actively dividing cells; especially useful for tumor therapy
 Single stranded RNA virus, DNA copy integrates into host genome
 Size limit 8 kb
 Vector replication and packaging functions are provided by engineered
cell line
 Problems: Recovery of replication competent retrovirus and low titer
Lentivirus (HIV) vectors:
 HIV causes lethal disease, safety concerns a disadvantage
 Single-stranded RNA virus, DNA copy integrates into host genome
 Infect proliferating and non-proliferating cells
 Can infect blood-forming (hematopoietic) stem cells
 Size limit of 8 kb
Herpes simplex virus:
 HSV causes many different diseases in humans, cytotoxic
 Double stranded DNA virus
 150 kb viral genome with 80 viral genes
 Capacity as a vector is about 30 kb
 Can infect a wide range of cells, including neurons
 Does not insert its DNA into host genome
b. non-viral
 Low immunogenicity; safe
 Easy to manufacture and store
 Inefficient transfection and short duration of expression
Naked DNA
 Works well for muscle, heart, skin
 DNA vaccinations can induce immune response against encoded antigens
 Not suitable for targeted therapies
 New methods allow injection into tissues without needles: "gene gun" or
Intraject
Cationic lipids or polymers




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Positively charged molecules that bind to negatively charged DNA
Protect DNA from degradation
Can be used to transfect cells in lung in cystic fibrosis treatments
Poor targeting
Inactivated in the blood
RNA-based therapies
 Antisense oligonucleotides
 Ribozyme (RNA enzymes)
 RNAi
4. Future prospects
Greatest limitations of current technologies:
 Small capacity of available vectors
 Targeting of vectors to particular cells or tissues
 Safety, especially cytotoxicity or immunogenicity—Death of Jesse
Gelsinger in 1999 in gene therapy trial with adenoviral vector for ornithine
transcarbamylase deficiency
 Need repeated doses for many of the types of gene therapy—in early trial,
one dose of adenovirus gene therapy for cystic fibrosis only lasted 2-4
weeks.
 Most strategies not widely applicable to many genes
Promising gene therapy concepts:
 Direct killing of tumor cells with genes delivered by Ad vectors. Ad vectors
may be most applicable for short term expression.
 Delivery of naked DNA for preventative vaccination against infectious
diseases.
 Naked DNA delivery of genes for cardiovascular disorders.
 AAV delivery for chronic single gene disorders such as hemophilia.
Human hereditary diseases:
SCID (severe combined immunodeficiency)
ADA 1990
 Retroviral vector used to deliver adenosine deaminase gene to Tlymphocytes of affected child
 Used with an enzyme therapy—how much of improvement due to gene
therapy?
X-linked SCID 2000
 Retroviral vector
 Deliver portion of the cytokine receptor gene IL2R
 9/11 patients were cured
 Complications: 2/11 patients developed leukemia, most likely due to
insertional activation of oncogene
Cystic Fibrosis
 Only 5-10% of normal CFTR levels needed to see improvement
 18 different clinical trials of therapies
 initial therapies used adenovirus—concern about immune response
 recent trials used liposomes or AAV
 problem in getting around mucus barrier in lungs and into correct subset of
glandular epithelial cells