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Gene Therapy


Recognize significance and uses/abuses of gene therapy. ... Good news: Promising advances during the last ... patient enrollment in study despite ineligibility. ... – PowerPoint PPT presentation

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Title: Gene Therapy

Gene Therapy
  • Objectives
  • Recognize significance and uses/abuses of gene
  • Define background theory and methodologies.
  • Address potential clinical applications,
    limitations, and future directions.

Paul Shapiro Room 536 PH
  • Goal of gene therapy
  • Management and correction of human diseases
  • a. Inherited and acquired disorders
  • b. cancer
  • c. AIDS/HIV

Good news Promising advances during the last two
decades in recombinant DNA technology. 1. Recent
success in treating SCID. Bad news (Until
recently?) Efficacy in any gene therapy protocol
not definitive. 1. Shortcomings in gene transfer
vectors. 2. Inadequate understanding of
biological interactions of vector and host.
(Jesse Gelsinger case).
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  • Some global terms/definitions
  • Genes and nucleic acids
  • 2. Vector, viral delivery systems
  • 3. Gene transfer
  • 4. Reporter gene
  • 5. Transfer efficiency
  • 6. Germ vs. somatic cells
  • 7. In/ex vivo, in situ, in vitro.

Genetic diseases Type 1 Single locus (gene)
is defective and responsible for the disease,
100 heritable. examples Sickle cell anemia,
Hypercholesterolemia Cystic fibrosis
Type 2 Polygenic traits, lt100 heritable, may
be dependent on environmental factors and
lifestyle. examples Heart disease Cancer D
iabetes Alcoholism Schizophrenia Criminal
behavior? etc.?
-Recombinant DNA technology 1940s DNA and not
proteins carried genetic information. 1950s
DNA structure 1960s - Restriction enzymes
and ligases 1970s - Cloning techniques,
detection, and sequencing. 1980s Polymerase
chain reaction 1990s - 1st successful gene
therapy protocol (William Anderson, Michael
Blaise, and Ken Culver) Treatment of ADA
(adenosine deaminase) deficiency-causes severe
immune deficiency. Recessive disease that
results in the buildup of waste products that
kill T-cells. Used retrovirus to infect
T-cells and produce ADA.
Gene Transfer
Exogenous DNA vector (viral)
Protein expression
Transcription (nucleases)
Barriers that prevent transfer of exogenous DNA
Vectors for gene transfer
Retroviruses promising candidates and widely
used. - Insert genetic material into host
DNA. - Insertion may disrupt a host gene. -
Insertion may be in a region that doesnt
produce very much of the desired protein - Can
trigger immune response.
  • Ideal vector characteristics
  • Insert size one or more genes.
  • Targeted limited to a cell type.
  • No immune response.
  • Stable not mutated.
  • Production easy to produce high concentrations
  • 6. Regulatable produce enough protein to
  • cause an effect.

Retrovirus life cycle
From Alberts et al. Molecular Biology of the Cell
Retrovirus genome
Encapsidation (packaging)
Retrovirus vector construction for gene therapy
5 LTR Packaging Gene X Neor 3 LTR
Retrovirus vector packaging for gene therapy
5 LTR Packaging Gene X Neor 3 LTR
Packaging cell line express viral gag and pol
Produce high titer recombinant virus in 24-72
Packaging cell line (makes virus)

Vector (transfection)
  • Collect virus after 24-72 hrs.
  • Concentrate virus / high titer.
  • Transduce host cell.

Summary of commonly used vectors
Retrovirus Adenovirus Adeno- Naked
DNA/ associated Liposomes
Insert size 8kb 30kb 4kb unlimited Integration
yes no rare rare Production gt106cfu/ml gt1011
gt1012 unlimited Administration ex vivo ex/in
vivo ex/in vivo ex/in vivo Expression long trans
ient pot. good? Transient Express level
moderate high moderate high Immune few extensive
?? None Safety concerns Insertional Inflammat
ory Inflammatory none? mutagenesis response
response toxic
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  • Non-viral DNA carriers
  • Cationic liposomes Positively charged lipids
  • with negatively charged DNA. (lipid-DNA
  • -Transverses cell membranes
  • Advantages
  • a. Stable complex
  • b. Can carry large sized DNA
  • c. Can target to specific cells
  • d. Does not induce immunological reactions.
  • Disadvantages
  • a. Low transfection efficiency
  • b. Transient expression
  • c. Inhibited by serum
  • d. Some cell toxicity

Non-viral DNA carriers
2. Naked plasmid DNA injection
Gene X
Promoter gene of interest (P)
(gene X)
Expression observed in thymus, skeletal and
cardiac muscle, skin.
Gene therapy to turn off genes Antisense
approach DNA makes mRNA, mRNA makes
protein. Antisense complements mRNA (sense)
and prevents protein expression. Small
interfering RNA (siRNA) molecules. Paper to
read The silent treatment siRNAs as small
molecule drugs. Ribozymes
Categories of clinical gene transfer protocols.
1. Inherited/monogenic disorders ADA
deficiency Alpha-1 antitrypsin Chronic
granulomatous disease Cystic fibrosis Familial
hypercholesterolemia Fanconi Anemia Gaucher
Disease Hunter syndrome Parkinsons 2.
Infectious Diseases HIV 3. Acquired
disorders peripheral artery disease Rheumatoid
Categories of clinical gene transfer protocols.
4. Cancer (by approach) Antisense Chemoprote
ction Immunotherapy ex vivo / in
vivo Thymidylate kinase Tumor suppressor
Case study Jesse Gelsinger
  • First documented patient to die from gene
    therapy treatment. (may have been others).
  • Disease liver enzyme deficiency
  • (ornithine transcarbamylase, OTC)
  • controls ammonia metabolism
  • Vector used to deliver OTC modified adenovirus
  • Goal deliver vector to liver cells and express
  • Problem Very low transfer efficiency (1),
    difficult to get
  • enough functioning OTC expressed to do any good.
  • Solution Infect with higher dose of viral
  • (38 trillion)

Outcome -Vector not only delivered gene to
liver but to other tissue. -Triggered systemic
inflammatory response. -Patient acquired fever,
coma, death. Why? -Animal studies suggested
dose was OK (?). -Adenoviral vectors known to
induce inflammatory response. -Patient already
compromised Patient had higher than allowed
ammonia levels.
Results of follow-up investigation
  • 3 month investigation by FDA concluded.
  • patient enrollment in study despite
  • participants misled on safety and toxicity
  • loosening of criteria for accepting volunteers.
  • informed consent document did not reveal results
  • of animal studies.
  • Other investigators may not have disclosed
  • information on patient deaths in gene therapy
  • Adenovirus safety Engineered to prevent viral
  • Mutation from replication incompetent to
  • Shut down of Univ. of Penn. Institute for Human
    Gene Therapy
  • Lawsuits

Some successes
  • Treatment of Severe Combined ImmunoDeficiency
  • Genetic defects cause decreased T and B cells and
    NK cells.
  • Affects 1-75,000 births.
  • Mostly males (most common form is X-linked)
  • Types ADA (adenine deaminase) or Gamma chain
  • Success in treating children observed in Italy,
    Israel, England, France, and USA.
  • Phase 1 trial collect bone marrow, isolate CD34
    stem cells, and infect with retroviral vector
    containing the gene encoding the g-common chain.
    Inject two infants with 14-26 million CD34
    cells/kg (5- 9 million contained the introduced

Recent successes continued
Phase I clinical trials results Detectable
levels of NK and T cells containing the
introduced gene were found in the blood within 30
and 60 days, respectively, and their numbers
increased progressively until normal levels were
reached. After 3 months, the two patients were
also able to make antibodies in response to
vaccination against diphtheria, tetanus, and
10-3-02 France and US (FDA) halted SCID gene
therapy due to leukemia-like side effects in one
child. Not clear whether this is related to the
gene therapy itself. 1/14/03 FDA suspended 30
gene therapy trials using retrovirus vectors due
to another case of leukemia.
Science. 2006 Oct 6314(5796)126-9.
Cancer regression in patients after transfer of
genetically engineered lymphocytes.
Lymph node
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