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13 Cards in this Set
- Front
- Back
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Gene Therapy
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-several steps
-production retroviral vector allowing entry therapeutic gene to patient` |
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Vector production
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-in vitro, directly inoculated into patient
-genetically modified blood cells from patient in vitro, re-inoculated hopes will proliferate |
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Ex vivo gene therapy
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-requires:
-vectors -ancillary factors -cells to target (autologous/allogeneic/xenogeneic) |
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Vectors
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-need to deliver efficiently in cells
-retroviruses -adenoviruses -adeno-associated viruses |
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Ancillary factors
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-cytokines
-growth factors -hormones |
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Target cells
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-fibroblasts
-heptocysts (for hepatocellular carcinoma) -hematopoeietic cells (easily removed and genetically modified) -keratinocytes -myoblasts -stem cells |
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Retro-vectors
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-needs essential info for functional virus: gal, pol, env, LTRs
-psi sequence: packaging RNA into genome -minimal: LTR, PBS, Psi, extended packaging sequence to ensure RNA incorporated to retroviral vector |
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proviral DNA gene therapy into tissues
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-minimal: LTR-psi-cDNA-LTR
-vector because cell its going into has gag, pol and env, its a packaging cell line |
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Packaging Cell Line
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-independently engineered so expresses gag, pol and env
-completely different genes cassettes or an integrated proviral DNA -transfect DNA into cell, gag/pol/env in cell help with repliaction cDNA construct and help to produce RNA -used to make a population of a virus to be purified and get retrovirus stock |
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Neo
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-selectable marker
-drug resistance, fluorescence -allows for selection of cells successfully expressing transgene -in vitro -used in sequences that express two genes |
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cDNA
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-therapeutic gene in sequences that express two genes
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Gene delivery
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-using retroviral vector
-CMV-RU5'-Psi-transgene-GFP-U3R -transfect into packaging cell line -integration occurs giving retroviral constrcut, transcribed to give mRNA spliced |
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Retrovirus stock
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-of replication defective retroviruses
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