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20 Cards in this Set
- Front
- Back
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What is the goal of gene therapy?
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Replace a defective gene by stably introducing a correct copy
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Advantage of gene therapy with RV?
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New gene will be permanently inserted into the host genome
Viral genome easily modified in vitro Simple strategy to produce infectious, non replicating particles |
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Disadvantages of using RV in gene therapy?
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Random integration site
May recombin with other RV |
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What is the general scheme of retroviral gene therapy
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1) Cells removed from patient
2) Virus altered in the lab so it can't reproduce 3) Insert a gene into the virus 4) Altered virus is mixed with cells from th patient 5) The cells from the patient becomes genetically altered 6) Altered cells are injected intothe patients 7) The genetically altered cells produce the desired protein or hormone |
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What are the 3 general constructs necssary for making a non-replicating retroviral vector?
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1) Transgene cassette" gene of interst incorporated into a retroviral vector, LTRs, PBS, Psi
2)Gag- Pol plasmid construct 3) Env plasmid construct 2 & 3 will be produced cuz they're placed in the transgene cassette |
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What are the strategies of gene therapy?
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-Apoptosis related gene
-p53 tumour suppressor Dominant negative ptn -Immunoregulatory ptn (cytokine, adhesion, MHC) -Toxin: Tk and gancyclovir is toxic to HSV replication and induces cell death in viral TK expressing cells. Also effective strategy in gene therapy for brain cancers |
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What is retroviral pseudotyping?
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Substitue retroviral ENV glycoptn with a dif ENV which encodes a viral receptor with a wider target host range (can infect more cells)
i.e. VSV G-ptn: surface ptn with a very broad host range, can infect just about any cell type |
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What are the limitations of gene therapy?
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1) Efficient delivery:
a) Ex vivo: remove cells from patient, ncubate with vector and return gene engineered cells to the patient b) In situ: vector is introduced directly into affected tissue: Ad vectors injected into bronchi of patients with CF. Dystrophin gene inmuscular dystrophy. Injection of tumour mass with vector carrying cytokine or toxin. c) In vivo: vector injected directly into the blood stream 2) Transduction of non-dividing cells: Most RV need cell division to replicate. In teh case of HIV, viral ptns can be injected after teh infection of a host cell 3) Long term gene expression: Robust expression when first introduced, but transgene tends to shut down after time, probably due to chromatin modification, acetylation... 4) Cost effective manufacturing |
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How does SCID come about?
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-Mutation in gamma common cytokine receptor chain
-Mutation in ADA |
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How did early gene therapy trial try to stop SCID? What were the results
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Introducing genes ex vivo in hematopoetic stem cells, then reinfused back into the patients.
-80% successful, had reconstituted immune system --many ppl dev'l leukemia (ATLL) -Why? Integration wasn't random. A quarter of time, integration within 10kb of the promoter. Preferential engrafment/survival of transformed cells with integration events near a proliferation gene |
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What is oncolytic virotherapy?
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-Cancer killing virotherapy
- Harness properties of viruses to fight cancers: have viruses replicate in cancer cells and leave normal cells unaffected -Has a high therapeutic index 1000:1 (much higher then chemotherpy 6:1) 0Has less side effects than chemotherapy -Initially tumour repression, but in the long term, tumours came back |
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What are the advantages of using VSV?
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Not a human pathogen
No neutralizing antibody Sensitive to host antiviral response Replicates only in teh cytoplasm No integration step Geneticall stable Rescue system easily manipulated by recombination techniques Grows at very high titer in GMP |
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What has VSV been used for?
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-Selectively targeted various cancers: breast, ovarian, malanoma, colon carcinoma, lung tumour derived cells and tissues
-> Tumour cell abnormalities provide a niche for VSV viruses (has innate immunity, antiviral program and .: can differentiate btw normal and tumour cells) -VSV can induce apoptosis of infected cells |
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Why does it take longer for epithelial infected cells to die?
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Slower dividing time
Pretreatment with interferon before adding VSV can protect normal cells |
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Why is VSV oncolytic?
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Mechanism unknown, but do know that VSV selectively infects and kills transformed cells. The IFN system is disrupted in transformed cells, .: it allows selective VSV oncolytic activity, while sparing normal non-transformed cells
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What are the mechanisms of Oncolysis?
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-Direct virus induced apoptosis: Virus enters, replicates and kills host cells. Normal cells have antiviral program, so cell death is limited
-Indirect apoptosis caused by inflammatory cytokine release: Body of tumour contains dying cells, which release cytokines which cause apotosis -Shutdown of tumour vasculature: selective, virus gains access to the tumour via neovasculatur, but also shuts down new blood cell formation -Adaptive immune reponse against tumour antigens: Cross-presentaton of tumour and viral antigens, not very well understood |
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How were the 4 heart-lung combinations from animals that were inoculated with CT26 (the carcinoma cells) treated?
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Ordinary animals were inoculated with colon carcinoma. Grape-like clusters of metastasized tissue for in their lung tissue.
1) Inoculate this with VSV and UV light so virus dies, metastasis is the same 2) Inoculate sampls withIntransal VSV introduces a dec in the amt of metastasized tissue 3) Multiple intranasal or intravenous injections diminish the presence of cancerous tissue to almost nothing -Survival curve for the animas highest with the last treatment -If animals re-expposed to CT26, they're unaffected, probably cuz of immune memory response |
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Whatwas the result of the time experiment with VSV and metatstatic nodes in the heart and lung?
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Conclusion of experiment was that VSV enters, multiplies quickly, has deadly effect for the cancer cells, but is self-limiting and shuts itself off
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What affect to histone acetylases and deacetylases (HAT and HDAC) have on genes?
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They have a role in the regulation of gene transcription through modulation of chromatin structure
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What are Histone Deacetylase Inhibiters (HDI)?
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-New type of molec tht have potent anti-tumour activity
-May sensitize cancer cells to the oncolytic effects of VSV by their capacity to inhibit residual innate response (IFN) in tumor cells -HDIs can be used as chemical switches to regulate cellular innate antiviral responses and to provide controlled growth of therapeutic viruses within malignancies -Inc oncolytic activity correlates to less cellular IFN responses and inc of virus induced apoptosis |
