Gene Therapy

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Gene Therapy
Paul Shapiro
Room 222
Objectives:
Importance and uses/abuses of gene therapy
Background theory and methodology
Clinical applications, limitations,
and future directions
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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:

1. 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.
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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, <100% heritable, may be dependent
on environmental factors and lifestyle.
examples: Heart disease
Cancer
Diabetes
Alcoholism
Schizophrenia
Criminal behavior
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Background:
-Recombinant DNA technology
1940s – DNA and not proteins carried genetic information.
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.
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Gene Transfer
Transcription
(nucleases)
Exogenous DNA
+ vector (viral)
Cytosol
Endosome
Lysosome
Protein expression
Barriers that prevent transfer of exogenous DNA
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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 doesn’t produce
very much of the desired protein
- Can trigger immune response.
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Ideal vector characteristics:

Insert size: one or more genes.
Targeted: limited to a cell type.
Immune response: none.
Stable: not mutated.
Production: easy to produce high concentrations
or titer.
6. Regulatable: produce enough protein to
make a difference.
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Retrovirus life cycle
From: Alberts et al. Molecular Biology of the Cell
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Retrovirus genome
5’ LTR Packaging Gene X Neor 3’ LTR
Encapsidation
(packaging)
Retrovirus vector construction for gene therapy
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5’ LTR Packaging Gene X Neor 3’ LTR
Retrovirus vector packaging for gene therapy
Packaging cell line express viral gag and pol
Produce high titer recombinant virus in 24-72 hours.
Packaging
cell line (293s)
1.
+
Vector (transfection)
Collect virus after 24-72 hrs.
Concentrate virus / high titer.
Transduce host cell.
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Summary of commonly used vectors
Retrovirus Adenovirus Adeno- Naked DNA/
associated Liposomes
Insert size 8kb ~30kb 4kb unlimited
Integration yes no rare rare
Production >106cfu/ml >1011 >1012 unlimited
Administration ex vivo ex/in vivo ex/in vivo ex/in vivo
Expression long transient pot. good? Transient
Express level moderate high moderate high
Immune few extensive ?? None
Safety
concerns: Insertional Inflammatory Inflammatory none
mutagenesis response, toxic response, toxic
(63%)
(16%)
(2%)
(13%)
(RAC approved)
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Non-viral DNA carriers:

Cationic liposomes: Positively charged lipids interact
with negatively charged DNA. (lipid-DNA complex).
-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

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Non-viral DNA carriers:
2. Naked plasmid DNA injection
cDNA
Promoter + gene of interest
(P) (gene X)
P
Gene X
Expression observed in thymus, skeletal and cardiac muscle, skin.
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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.
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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 arthritis
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Categories of clinical gene transfer protocols.
4. Cancer (by approach):
Antisense
Chemoprotection
Immunotherapy: ex vivo / in vivo
Thymidylate kinase
Tumor suppressor genes

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Case study: Jesse Gelsinger
*First 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 OTC.

Problem: Very low transfer efficiency (1%), difficult to get
enough functioning OTC expressed to do any good.

Solution: Infect with higher dose of viral particles (38 trillion).
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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.
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Results of follow-up investigation:
3 month investigation by FDA concluded.
patient enrollment in study despite ineligibility.
participants misled on safety and toxicity issues.
loosening of criteria for accepting volunteers.
informed consent document did not reveal results
of animal studies.
* Other investigators may not have disclosed important
information on patient deaths in gene therapy trials.

Adenovirus safety: Engineered to prevent viral replication.
Mutation from replication incompetent to competent?
Shut down of Univ. of Penn. Institute for Human Gene Therapy
Lawsuits
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Recent successes:
Treatment of Severe Combined ImmunoDeficiency (SCID)
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 (gc).
Success in treating children observed in Italy, Israel, England, France, and USA.

The phase 1 trial involved harvesting the patient`s bone marrow, isolating the CD34+ stem cells, and infecting them with the retroviral vector containing the gene encoding the g-common chain. At the end of the procedure, the two infants received 14 million to 26 million CD34+ cells/kg, of which 5 million to 9 million contained the introduced gene.
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Recently (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.
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 pertussis.
Recent successes continued: