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26 Cards in this Set
- Front
- Back
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Ex vivo |
Target cells removed from host, altered in VITRO and reintroduced into host |
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In vivo |
Target cells altered while still in the host |
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Germline gene therapy |
Altering DNA that is passed onto offspring, illegal |
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Somatic Gene therapy |
Altering DNA that is not passed to offspring, legal |
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4 classifications of gene therapies ("RASE"), describe each |
Replacement: Replace or subsititute a defective or missing gene (monogenic diseases) Addition: Adding a new or changed function to an exisiting cell (HIV resistance or B cell to attack tumors) Silencing: Arresting or antagonizing an undesired gene (viral or oncogens) Editing: Removing or correcting deleterious material (CRSPR or Zinc Finger) |
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Problems of Naked DNA (4) |
1.) Rapidly cleared from circulation 2.) Degraded in vivo by endonucleases 3.) Cannot enter cells on its own 4.) Degraded in the cytoplasm and cannot enter the nucleus by itself |
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Advantages of Viral Vectors (3 in general) |
1.) High efficiency in gene delivery 2.) High protein Production 3.) May achieve long term transgene expression |
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Disadvantages of Viral Vectors (5 in general) |
1.) Limit on insertion length of transgene 2.) May cause severe immunogenicity (especially upon second injection) 3.) May cause insertional mutagenesis (retroviruses) 4.) May integrate w/ wild type viruses adn become virulent 5.) Large scale production remains a challenge |
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Advantages of Non-Viral vectors (4 in general) |
1.) Safe, non infective 2.) Can carry large transgene segments 3.) Easy for large scale production 4.) Non- immunogenic |
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Disadvantages of Non-Viral Vectors (3 in general) |
1.) Low gene delivery efficiency 2.) Low transgene expression level 3.) Only transient/short term gene expression |
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What gene delivery vectors are used most in RCTs currently? |
#1= Adenovirus #2= Retro Viruses |
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Ideal components of Non viral vectors (4) |
1.) DNA Condesing Agent 2.) Targeting Moiety 3.) Endosomolytic agent 4.) Nuclear localization sequence |
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Describe 2 Mechanical methods and 4 Physical methods of gene transfer (used IN or EX vivo?) |
Mechanical: Microinjection, Gene gun Physical: Electroporation, sonoporation, laser irradiation, magnetofaction |
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Describe DNA vaccines, their MOA, ROA, and advantages |
MOA: Plasmid w/ cDNA for antigens injected directly into host muscle or skin cell, transfect cells, antigen proteins produced = Humoral, cell mediated, and mucosal immunity ROA: IM or SQ PROS: Cheap, stable, safe, provides strong immunity |
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Indications, MOA, ROA and vector of: Gendicine |
Indications: Head and Neck cancer MOA: Inserts p53 gene into p53 deficient (tumor) cells, leading to decreased proliferation and cell apoptosis ROA: Intra tumor injection Vector: Adenovirus5-p53 (replication deficient) |
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Indications, MOA, ROA and vector of: Oncorine |
Indications: Head and Neck cancer MOA: Replication competent Ad vector w/ deletion in gene responsible for inactivating p53 may only replicate in p53 deficient cells (tumor cells). Preferential replication in tumor cells leads to cell deal via lysis (release of viral vectors) ROA: IV Vector: Adenovirus w/ deletion in E1b gene |
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Indications, MOA, ROA and vector of: Neovasculogen |
Indications: PAD and Critical Limb Ischemia MOA: Insertion of transgene that codes for VEGF= vasodilation and proliferation ROA: IM Vector: pDNA for VEGF |
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Indications, MOA, ROA and vector of: Glybera |
Indications: Lipoprotein LIpase Deficiency (LPLD) a monogenic disease MOA: rAAV = transgene expression in muscle cells of lipoprotein lipase enzyme ROA: One time, multiple IM injections (into calf) Vector: rAAV ($$) |
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Indications, MOA, ROA and vector of: Imlygic (Talimogene laherparepvec or T-VEC) |
Indicaitons: Melenoma (skin and lymph nodes) MOA: Only replicates in TUMOR cells, leads to secretion of GM-CSF (cytokine)= immuno attack on tumor cells and oncolytic replication ROA: Intra tumor inj Vector: HSV coding for GM-CSF (Cytokine) and only capable of replicating in tumor cells (enzyme deletion) |
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Antisense Oligonucleotide (AONs): General MOA, Pros and Cons |
MOA: Short 18-21NT sequences w/ perfect base paring to target sequence that bind mRNA, DNA or non-coding RNA and inhibit its transcription or translation PROs: Easy to make, long term, sensitive, specific Cons: Stability, renal excretion, short t1/2, limited cellular uptake, potentila for off target effect (BAD) |
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Vitravene/Fomivirsen: Indication and MOA |
Indication: AIDS related Retinitis MOA: AON |
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Kynamro/Mipomersen: Indication and MOA |
Indication: Homozygous Familial hypercholesterolymia MOA: AON |
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CONS of AONs/RNAis (delivery |
Fast Renal elimination Limited cellular uptake Immune responses Subject to nucleases ONLY able to ANTAGONIZE TARGETS |
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Barriers to efficient AON/RNAi delivery |
Strong Plasma protein binding Penetration of cell membrane and cel nucleus Escape from endosomal vesicles |
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Explain the process of miRNA interference |
Pri-miRNA produced from intron DROSHA processes into Pre-miRNA pre-miRNA transported out of nucleus binds w/ DICER produced mi-RNA mi-RNA associates w/ argonaut = RISC Guide strand kept, RISC binds sequence complementary to miRNA= translational repression or target degredation |
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Explain the process of siRNA interferance |
Exogenous siRNA endocytosized siRNA escapes endosome siRNA associates w/ argonaut = RISC RISC keeps guide strand Complex binds complementary to siRNA=mRNA degredation |
