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65 Cards in this Set
- Front
- Back
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What are the ways to prevent or control viral diseases?
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Vaccines
Antiviral drugs: small molec that block viral replication Antiviral gene thera[y |
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What are the different kinds of antiviral gene therapies?
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Ptn based
DNA or RNA RNA |
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What are ptn based antiviral gene therapies?
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Transdominant inihibitors
Intracellular Abs Capsid-nuclease hybrid ptns Inducible toxins or killer ptns |
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What are some DNA or RNA based antiviral gene therapies?
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Antisense oligonucleotides
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What are some RNA based antiviral gene therapies?
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RNA decoys
Ribozymes Small interfering RNAs |
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What are RNA decoys?
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RNA molec that mimic natural viral RNA structures
these structures are produced in large amts in the cell and will compete for the cellular and viral factors bound to it ->These factors will become unavailable for viral expression and replication |
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For which viruses have RNA decoys been used against?
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HIV
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What was used as RNA decoys vs HIV?
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TAR structures
Psi structures RRE structures |
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How does an RNA decoy work?
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A viral or cellular ptn bing to the structural RNA of the virus
These compete out the native structure that usually bind the virus |
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Which RNA decoy is in clinical trials for HIV?
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TAR RNA
(RRE RNA also exists, but not in trials) |
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What is TAR RNA coupled with?
Why? |
U16 snoRNA
Used to improve efficacy and make it more active |
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What does HIV TAR decoy coupled to U16 snoRNA do?
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Decreases HIV replication in lymphocytes
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Where is this expressed?
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Expressed on a retroviral vector that uses a U6 promoter (uses RNA pol so RNA not cut)
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What are antisense oligonucleotides?
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DNA or RNA molec that hybridize with an mRNA and inhibit its fct
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What are the 2 mechanisms through which this can work?
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1) RNA degradation due to cleavage by cellular RNAse H (cleaves antisense DNA, if DNA-RNA hybrid) or by RNAse1 (if RNA-RNA hybrid)
2) Tl inhibition (steric antisense oligonucleotides) |
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When did antisense methods start?
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20 years ago
->Every pathogenic virus has been targeted |
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What have been important developments in antisense methods?
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Addition of a chemical cmpd (steric)
Combination with targets of various RNAses (RNAseP and RNAse L) |
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What can the antisense oligo method be combined with?
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Ribozymes
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How do antisense oligos inhibit Tl?
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either ssDNA or ssRNA bind the mRNA, don't allow Tl machiner y to bind
They don't come off .: get degradation of it by RNAse H (if DNA-RNA) or RNAse L (if RNA-RNA) |
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What else can steric antisense oligs use?
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Can bind a chemical gp to block Tl
->Recent targets not enough to completely inhibit viral replication |
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What is a ribozyme?
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An ENZ that uses RNA (not ptns) to carry out catalytic activity
->Catalytic RNAs that cleave themselves in their natural form (selfcleavage) |
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What can ribozymes be designed to do?
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Specifically pair with and cleave a specific target in trans (trans cleavage)
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What do naturally existing catalytic RNAs include?
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Hammerhead
Hairpin Hepatitis delta virus RNA Gp I and II introns RNA subunit of RNAse P |
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What can be done to increase the specificity of the ribozymes?
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Ribozyme "catalytic centers" have been incorporated into antisense RNA
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What are some small ribozymes?
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Hammerhead and hairpin
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What do these do?
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Can make a stranf that will oligomerize and cleave the target RNA
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What seq does the target have to have for hammerhead or hairpin to cleave?
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NUH seq
N= any nuc H= C/U/A .:need to have: N--U--C/U/A |
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What are some large ribozymes?
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RNA moiety of RNAse P
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What is RNAse P?
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Endonuclease that generates the mature ends of tRNAs
In bacterial RNAse P, the RNA subunit (RNAse P Rz) has catalytic activity and the ptn component facilitates the Rz-substrate binding |
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Describe the Hep D delta ribozymes
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Only example of a ribozyme derived from a RNA species found in human cells
Has the natural ability to fct in the presence of human ptns Results in increased stability Cleavage capacity is intrinsic to the life cycle of HDV |
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How do you design a ribozyme?
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Design it so that it is homologous to what you want to cleave so that you can cleave another RNA in trans
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What happens once the RNA is cleaved by a ribozyme?
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It will be degraded by host nucleases
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What are some viruses that have been targetted by ribozymes (Rz)?
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HIV
HHV HCV HBV Influenza A HPV |
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What is the new generation of HDV Rz?
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The SOFA Rz
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How does an HDV Rz work?
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Cleaves well in vitro
Only needs 7 nuc to bp to gget cleavage But don't want to target the host and with only 7 nuc required to cleave, have a high chance of cleaving off target |
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What does the SOFA Rz have that previous HDV Rz didn't have?
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SOFA is a Rz that is combine with a biosensore (antisense olig)
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How does the SOFA Rz work?
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Rz binds and the biosensor (olig) also binds the target RNA
If not in the right conformation, the Rz won't be able to cleave because bound to BS (.: won;'t be able to cleave host RNPs) |
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What is the job of the biosensor (BS)?
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Blocks Rz if target isn;t there
Doesn't block if the right RNA is present |
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What needs to be designed for this SOFA Rz to work?
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Need to design:
P1 stem Blocker linker stem (btw BS and Rz) BS: homologous to target RNA |
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How did Rz work vs HIV?
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Used a Rz that was common to a conserved region of all 3 RNAs (9,4 and 2 kb)
It inhibitted 80%, but still needed to be improved |
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What are small interfering RNAs?
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Cause RNA interference
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What is RNA interference?
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Natural mechanism used by eukaryotes for gene silencing
Mechanism uses dsRNA identical to a target gene that will degrade its RNA |
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What happened when long dsRNA was used?
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Didn't work in mammalian cell because it activated IS
Would be degraded immediately through PKR action, which shuts down cellular and viral Tl |
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What happens if cells are transfected with an exogenous dsRNA homologous to a target mRNA?
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The target mRNA is degraded
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Do small dsRNAs have the same effect as llong dsRNA?
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No, small dsRNA (21-23nucs) can inactivate gene expression adn do NOT activate PKR in mammalian cells
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How can small dsRNAs be introduced?
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Can be transfected into cells as small interfering RNAs (siRNAs)
Can be produced on transfected vectors: short hairpin (shRNAs) |
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Do natural small regulatory RNAs exist in the cell?
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Yes--> microRNAs (miRNAs)
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Do siRNAs have activity in vitro?
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No, need ptns from cells
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What ptns are involved in RNA interference?
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Dicer
Ptns from Argonaute family dsRNA binding ptn |
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What does Dicer do?
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Belongs to RNAse III family
Mammals only have 1 dicer: cleaves long dsRNA to generate siRNA/miRNAs Drosophilas have 2 dicers (dcr1/2) |
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What ptn from the argonaute family is required in mammals for siRNA?
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Ago2
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What does Ago2 do?
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Ago2 binds Dicer
Ago2 cleaves the RNA hybridized with one siRNA |
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What does the dsRNA binding ptn do?
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Binds to Dicer and is required for Dicer and Ago2 fct
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What is the dsRNA binding ptn in mammals?
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TRBP (TAR RNA binding ptns)
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What complex is formed by TRBP-Dicer-Ago2?
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RNA-induced silencing complex (RISC)
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What is the mechanism of RNA interference if it follows the exogenous path?
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1) Virus/transposon/exogenous siRNA enter the cell and are bound by Dicer and TRBP (+ other ptns)
2) dsRNA/shRNA are cleaved by Dicer to make siRNA 3) RISC complex (TRBP/Dicer/Ago2) bind siRNA. Get strand separation 4) One strand will bind to homologous RNA target. Ago2 will mediate the cleavage of the RNa bound to the siRNA 5) Get mRNA cleavage |
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What do you do when you design your siRNA?
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Make it perfectly homologous to the target (if you have a mismatch, won't work as well)
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What happens in the endogenous path?
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1) miRNA is T from the genome
2) Formed as pri-miRNA in the nucleus. Bound by drosha: mediate cleavage in nuc and DGCR8. Form precursor of miRNA, which leaves the nucleus 3) When pre-miRNA reaches the cytoplasm, it is bound by TRBP and Dicer ->In natural path, always have mismatches, never perfectly homologous binding 4) Dicer cleaves. Get RISC formation and strand separation 5) One strand binds target RNA --> Mainly works through Tl repression (very little mRNA degradation) |
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What does the exogenous pathway usually result in?
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mRNA degradation
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What are the adv of using siRNAs?
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Easy to design
Very effective |
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What are the limitations of using siRNA?
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Need an exact match
Viral escape (see inhibition at first, but after a while, the viral load inc) May induce IFN path and PKR activation Delivery Limited amt: can't use too many because oversaturate cellular factors and won't be available for cellular endogenous needs) |
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What are different methods of delivery?
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Transfection (can't rly be done in vivo)
Intranasal/tracheal (only for resp viruses) Hydrodynamic injection (no good for humans) Liposomes Viral vector for gene therapy |
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What are alternatives for siRNAs?
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Target cellular genes required for replication
Adapt the delivery method to the virus ans route of infection |
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What are clinical trials in progress using RNA-based technologies?
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siRNA vs respiratory viruses (RSV, parainfluenza and influenza virus): Phase I for intranasal use
Decoy.antisense, Rz, siRNAs vs HIV: by gene therapy (Phase I/II) -->Being done with retroviral or lentiviral vectors -11 in lymphocytic T cells and 6 in hematopooietic stem cells -One triple combo with siRNAs vs Tat/Rev + nucleolar TAR decoy + anti-CCR5 Rz (phase 2) -1 completed phase 3 with hammerhead Rz: safe but not enough for long term inhibition |
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What is the future of this?
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Combination therapies vs viral and cellular targets
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