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25 Cards in this Set
- Front
- Back
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gene therapy: What is it ?
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Identification, isolation and repair of naturally occurring genetic mutations in
human alleles ... |
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Where did the idea arise ?
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Proposed as a possibility shortly after the discovery of genes (Science Fiction)
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Why bother ?
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Unlike treatment options, gene therapy has the potential to cure a
disease by repairing the source of the problem... Note: Gene therapy research is well-funded by governments and largely ignored by pharmaceutical corporations |
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Has it worked ?
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Gene Therapy has been successfully applied in (very) limited clinical settings
(ie. hemophilia, diabetes) |
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In principle, any disease can be targeted for gene therapy.
In practice, we need to know the following: |
Does the disease arise from a mutation in one (or a few) genes?
(5)* Will a normal gene solve the problem (dominant negative mutations)? (6)* Can we deliver a normal gene to the affected tissue? At present, diseases that result from a point mutation in a single gene that affect easily accessible tissues (ie. epidermus, blood) are the best candidates for gene therapy |
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Hereditary Diseases are generally divided into ______ and _______
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single gene; polygenetic
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Distinguish Dominant from Recessive in terms of mutation and disease
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- Dominant mutations only require a single mutant allele to cause a phenotype
- Recessive mutations require mutations in both alleles for a phenotype individuals with a single mutant allele (carriers) are healthy but may pass the disease to their progeny |
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Describe Linkage pattern in terms of mutation and disease
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a) Autosomal (non-sex linked) – typically follow normal Mendelian inheritance
pattern b) X-linked – fathers always pass to daughters; never pass to sons: mothers may pass mutation to sons and daughters (50%) c) Y-linked – fathers always pass to sons d) Mitochondrial – maternal inheritance; always passed from mother to progeny |
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give an example of an Autosomal Dominant disease
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Huntington's Disease (neurological disorder)
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give an example of an Autosomal Recessive disease
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Cystic Fibrosis (breathing and digestion), Tay-
Sachs (impaired ganglioside degradation), Sickle Cell Anemia (hemeglobin polymerization) |
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give an example of an X-linked Dominant disease
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Aicardi Syndrome (brain development)
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give an example of an X-linked Recessive disease
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Muscular Dystrophy (muscle wasting and more),
Color Blindness, Hemophilia A |
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give an example of a Y-linked disease
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male infertility
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give an example of a Mitochondrial single gene disease
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Leber's Hereditary Optical Neuropathy (loss of central
vision) |
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Why are Polygenetic Diseases a poor choice for gene therapy?
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Hereditary Diseases associated with multiple genes, lifestyle and
environmental factors Poor choice for gene therapy as require knowledge of all genes involved - Environmental and lifestyle factors make identification of genetic mutations difficult - Inheritance patterns are complex and non-Mendelian (tend to run in families but largely unpredictable) Examples include autism, hypertension, obesity, cleft palate some forms of cancer, diabetes and heart diseases |
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Identification of mutant gene(s) is largely dependent upon________?
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determining
inheritance patterns: Still need to verify mutation gives rise to disease Techniques: homologous recombination, gene silencing, deletion Problem: must use model systems – tissue cultures and animal models |
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Large eucaryotic genes are still non-trivial to clone (due to processing)... why?
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Involves generating many copies of gene (PCR) and transferring gene to an
organism that rapidly replicates (eg. bacteria, virus, yeast, insect) |
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Name and describe 3 means of Detecting Gene Expression??
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(1) Western Blot – use of specific antibodies to detect presence of
gene product in a given tissue type Requires purified protein sample to raise antibodies (2) Transcriptomics – use of complimentary mRNA array (gene chip) to detect presence of specific gene transcription Requires complete set of complimentary mRNA (3) Proteomics – separation of cellular protein followed by protein Mass Spectroscopy based identification or fingerprinting Requires complete set of protein sequence information |
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in terms of gene expression, there are 3 types categorized by their distribution... what are they?
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(1) Housekeeping – genes expressed in many or all tissue types
Associated with essential cellular functions (transcription, translation, replication, cellular metabolism) (2) Tissue Specific – genes expressed in a single or few tissue types Associated with differences between tissue types (3) Developmental – genes temporally expressed at specific times during life cycle Tissue differentiation, sexual maturity Note: Tissue specific and developmental gene may be secreted |
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_______ expressed in a single tissue type are the best _________ targets
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Secreted genes;gene
therapy |
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A repaired gene is often but not always sufficient to repair function... what are 3 reasons for this?
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(1) Dominant negative mutations
Mutant gene product may interfere with normal gene product function eg. both mutant and normal protein can bind to target but only the normal protein can stimulate target to function (2) Disrupted regulation Presence of multiple gene copies (mutant and normal) may prevent the production of sufficient quantities of normal gene product (3) Unwanted activity Mutant gene products may produce unwanted metabolites that are ultimately responsible for disease |
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Gene delivery is the Key to successful gene therapy
Successful gene delivery involves: |
(1) Targeting: repaired gene must specifically enter the correct cells
(2) Activation: repaired gene needs to enter nucleus and be successfully transcribed in response to regulatory cues (3) Integration: for long term protection, the repaired gene may need to integrate into the genome and be replicated (4) No side effects: the introduction of any foreign biological material runs the risk of being toxic, damaging the cells or stimulating an immune response |
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There are 3 approaches to gene delivery (delivery vectors) - briefly describe them
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Ex vivo – Tissue is removed, repaired gene is delivered and tissue is returned
Pros – highly specific, can test for activity and integration Cons – cannot introduce repaired gene into all cells of a given tissue; Only useful for secreted gene products – generally successful In vivo – Viral vectors Pros – Good at targeting specific cell types and integrating into DNA; can be modified so they do not replicate and destroy cell Cons – Size limitations, Side effects (Immune response) In vivo – Non-viral vectors (closed, circular DNA) Pros – No size limits, no immune response Cons – Less specific, less efficient integration Can be used for all types of gene therapy |
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What are some new approaches in gene therapy?
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Repairing Genes
Splicesome Mediated RNA trans-splicing Introduce gene that produces RNA designed to (1) block normal splicing reaction to mutant exon (2) promote splicing to repaired exon Inactivate Mutant Genes (dominant negative fix) Antisense RNA – Introduce gene that produces RNA complementary to mutant gene mRNA Forms an RNA duplex that cannot be translated Triple Helix – Introduce DNA that specifically binds to major groove of mutant gene and prevents transcription Ribozymes – Introduce gene that produces catalytic RNA targeting mutant gene mRNA |
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How is gene therapy like today's science fiction?
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Gene therapy was viewed as Science Fiction 40 years ago when the
genes were shown to be basic units of heredity Gene therapy is now a reality and there are many research programs aimed at making gene therapy approaches widely applicable (still years to go ...) So what can we look forward to (be afraid of?) in another 40 years .... Application of Gene Therapy approaches to germline “Designer” progeny – currently being proposed as means to prevent genetic diseases; short step to incorporating “beneficial” genes associated with increased lifespan, resistance, etc Cellular Repair Primarily targeting age related accumulation of mutations |
