MEDG520
Block 2
Gene Therapy
Major Concepts:
 What is gene transfer? gene therapy? a transgene? … a vector?
 What is a viral vector?
 What is non-viral gene therapy?
 What broad types of diseases have been treated with gene therapy?
 Where are the two main routes of gene delivery?
 Strategies to replace a loss-of-function mutation
 What is a loss-of-function mutation?
 Strategies to inhibit a dominant gain-of-function mutation
 What is a gain-of-function mutation?
 What are the advantages of gene therapies?
 What are some current limitations of gene therapies?
 What is the single most optimal vector for all gene therapies?
Minor Concepts:
 Recessive and Dominant Inheritance
 Promoter
 RT-PCR
 Immunohistochemistry
 Types of Mutations
 Reporter Gene
 Mouse model of human disease
 Large animal models of human disease
What is gene transfer? gene therapy? a transgene? … a vector?
What is a viral vector?
Vector
First generation
adenovirus (Ad)
Helper Dependent Adeno
(HD-Ad)
Adeno-Associated Virus
(AAV)
Lentvirus
Retrovirus
Capacity Advantages/Disadvantages
~7 kb
Short expression, toxicity, viral genes on vector cause these
problems. Easy to make.
~36 kb
Long term, a little toxicity from residual virus. Difficult to purify
from helper virus.
~4.5 kb Very long term (>4 yrs), no viral genes. Not simple to produce,
but promising.
~8 kb
Long term, possible toxicity, infects non-dividing cells.
~8 kb
Long term, low levels of expression, high potential for insertional
mutagenesis.
What is non-viral gene therapy?
 Naked DNA - large transgene capacity, short term, low levels of expression
 Cationic lipid mixed with DNA - large transgene capacity, short term.
 Electroporation - relatively large transgene capcity, short term.

Physical injection into cells (ie. gene gun, etc.) - large transgene capcity, short term.
What broad types of diseases have been treated with gene therapy?
1. Genetic diseases
a. Restore normal gene for loss-of-function mutations
b. Inhibit gain of function mutations
2. Cancer (Acquired genetic disease)
a. Oncogene inhibition
b. Restoration of tumour suppressor genes
c. Tumour cell-killing
d. Immunomodulation: increase immune response to tumours
e. Anti-angiogenesis (block formation of blood vessels to tumours)
3. Infectious diseases
a. Inhibition of viral genes, viral decoys…
b. DNA -based vaccination (DNA vaccines)
4. Other:
a. Cardiovascular disease (ie. Block restenosis after angioplasty, Angiogenesis)
b. Organ transplantation (engineer transplant to produce immunosuppressive antibodies),
c. “Gene medicines”
Where are the two main routes of gene delivery?
1. In vivo - Gene transfer occurs in the patient (non-viral vectors, viral vectors)
2. Ex vivo - Gene transfer occurs outside the patient
a. Autologous cell modification (from patient) and re-introduction (ie. electroporation,
transfection, viral, and non-viral vectors)
b. Non-autologous cell modification and implantation
(ie. encapsulation and implantation of a genetically engineered
cell line not derived from the patient)
Strategies to replace a loss-of-function mutation
 eg. gene therapies for Hemophilia
What is a loss-of-function mutation?
Strategies to inhibit a dominant gain-of-function mutation?
1. RNAi against mRNA of gene to knock down.
2. Antisense against mRNA of gene to knock down (ie. antisense Her2 gene).
3. Ribozyme against mRNA of gene to knock down (ie. anti-Her2 catalytic ribozyme).
4. Antibody against protein to knock down (ie. Herceptin).
5. Natural inhibitors of protein/mRNA to knock down (ie. Herstatin).
What is a gain-of-function mutation?
What are the advantages of gene therapies?
1.
2.
3.
4.
5.
Treatment of a genetic disease at the root of the problem, at the DNA level.
Potential to treat a disease for which no treatment is currently available.
Potential for life-long treatment from a single injection.
The patients own body produces the therapeutic gene product.
Once a treatment for one genetic disease has been developed, similar diseases should be equally
treatable, using a different disease-specific gene.
What are some current limitations of gene therapies?
1. Immune response to the initial administration of a viral vector, so that only a single-application of
a certain vector type would be possible because antibodies against that vector become so high that
a second administration is ineffective.
a. Methods to overcome include using alternate viral serotypes and transient
immunosuppression at the time of vector administration
2. Immune response to leaky expression of viral vector genes (transient expression). For example,
the first generation adenovirus produces low levels of “late” viral genes (late viral genes includes
gene like the capsid proteins, and these are highly immunogenic).
a. Overcome with the development of gutless viral vectors (ie. AAV, HD-Ad). ie. AAV does
not contain any viral genes, so there is no “low level viral gene expression”.
3. Immune response to the “foreign” transgene in null allele patients is a large obstacle: Attempts
have now been made to overcome with:
a. Choice of patients (ie. Select patients that contain specific mutations that produce
catalytically defective protein, ie. for Lipoprotein lipase deficiency, 80% of patients
produce immunoreactive protein, but they do not function normally).
b. Choice of vector (ie. AAV less immunogenic than Adenovirus)
c. Choice of injection site (ie. Liver less immunogenic than muscle)
d. Choice of promotor (ie. CMV versus a tissue specific promotor so the transgene is
expressed in antigen presenting cells)
e. Immunosuppression (Transient or long-term)
f. Use of non-foreign genes (ie. genes with similar function ie. utrophin instead of dystrophin
for the treatment of DMD, or the use of embryonic genes that have the same function but
are no longer expressed at high levels)
g. In utero treatment before the immune system fully develops
h. Germ-line gene therapy: in debate
What is the single most optimal vector for all gene therapies?
 Currently, there is no optimal gene therapy vector, different sites on the body that require the gene
and different requirements for durations of treatment require different vectors. For example, the
treatment of cancer requires short-term efficacy, just enough to kill the cancer cells; whereas,
treatment of Huntonton’s disease would likely require life-long gene expression from a single
injection. In addition, vector safety is now a significantly more important issue than it has been in
the past.
Recessive and Dominant Inheritance
See concept in Gene Discovery
Promoter

Region of DNA to which RNA polymerase binds before initiating the transcription of DNA into
RNA. Factors that regulate gene transcription do so by binding at or near the promoter and
affecting the initiation of transcription.
RT-PCR
 PCR amplification of mRNA used to detect transcription of a gene
Immunohistochemistry
 Method to detect in situ translation of a gene by detecting a specific protein in a tissue section
using a specific antibody against the protein.
Types of Mutations
 insertion, deletion, frameshift, missense (substitution), silent, nonsense (early stop), splice-site.
Reporter Gene
 ie. GFP or B-Galactosidase for which detection is simple and accurate
Mouse model of human disease
 ie. Hemophilia mice. Generally used for initial research depending on the specific animal model.
Large animal models of human disease
 ie. Hemophilic dogs. Generally used for pre-clinical research in the final stages before human
application.