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RNA Technology by Dr.Daoud and Nurse Kantaris
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Terms in this set (70)
how many nucleotides make up an oligonucleotides?
how many bases?
20 nucleotides
(or 20 mer, mer just means number of nucleotides)
generally 100 or less bases.
what is the function of oligonucleotides?
They design and synthesize complementary nucleotides to specific sequence of
DNA or mRNA
where do oligonucleotides usually accumulate?
they accumulate in cells by receptor mediated endocytosis
(which is not very efficient)
how can we efficiently cause oligonucleotides to accumelate in cells?
we use microinjection or liposomes (a lipid carrier that crosses the cell membranes)
what usually degrades the Oligonucleotides in the cell?
how do we avoid that?
They are usually degraded by intracellular nucleases in the cell
There is current research to modify them to avoid degradation
what is the earliest modification that usually happens to oligonucleotides?
how does that help?
Phospho sulfur backbone modification
1)Improves resistance to nucleolytic degradation
and
2) increases affinity for the plasma membrane to hinder clearance of Oligonucleotides
Do modifications usually increase lipophilicity or Hydrophilicity?
(remember, we want them to be excreted less and to be stuck to the plasma more)
increases Lipophilicity
where do the substitutions for ONs usually happen?
how does that help?
at the 2' position of sugar, sometimes 3'
this improves potency, stability and overall PK and PD properties
what are the antisense ONs?
what do they do?
they are antisense molecules that block steps in between the process of going from
DNA--->proteins
they basically fuk with RNAs before they can make proteins
what are the mechanisms that antisense molecules may interact with pre-mRNA in the
they interact with pre-mRNA in the nucleus
They cause
RNAse-mediated degradation
of target mRNA
Mask ribosome binding site on RNA
cross-link to target mRNA
Alter mRNA splicing
Inhibit endogenous miRNA
what is miravirsen (a drug)
an LNA that inhibits microRNA-122 (treats hep C)
What is mir-122
a liver specific mRNA required for infection of Hepatitis C virus (HCV)
so basically what's the whole point of creating an antisense pair?
how does it prevent protein synthesis?
what usually happens:
normal gene creates a messenger RNA which then synthesises a protein
What happens with antisense:
we create an antisense gene, that creates an antisense RNA
the antisense RNA binds to the Messenger RNA , it won't be available to create proteins anymore.
when you design an antisense, is it going to be there forever?
nope, it will eventually be degraded by RNAse H (a nuclease)
what are the applications of Antisense technology?
inhibition of dangerous or malfunctioning genes in microbes or other cellular processes
This can replace ACE inhibitors other therapeutic agents that usually target proteins, because
we can start targeting mRNA and DNA instead.
what are diseases that are a target for this method?
HIV, CML, and oncogenes
what is an example of Antisense tech that has been done?
The Flavr Savr tomato ,
a tomato that has an antisense mrna that inhibits polygalacturonase orPG
(the gene that is responsible for tomatoes ripening)
how does antisense therapy help?
Therapy that uses antisense oligonucleotide, antisense RNA or antisense DNA to target mRNA of normal or mutated gene
are antisense RNAs always made synthetically? or are they present endogenously sometimes?
They can be endogenous
some microbes, plants, animals make antisense RNA to regulate their own therapy
what are examples of antisense therapy?
Cancer specific antisense therapies like
Genasense
,
(In phase III for late-stage malignant melanoma)
vitravene for Cytomegalovirus or CMV (pulled from market
how does Genasense work
in clinical trials for cancers of liver, pancreas, colon, breast, kidney and esophagus
it targets and block expression of bcl-2
What is the bcl-2 gene?
a protein that blocks apoptosis,
without apoptosis you get uncontrolled proliferation
it is also implicated in resistance to chemo
TOPIC #2
what is RNA interference?
(RNAi)
discovered in 1998
worms were either fed or
injected with dsRNA
Resulted in inhibition of protein synthesis
what does RNAi cause ?
Post-Translational gene silencing (PTGS)
inhibits protein synthesis
using double stranded RNA (dsRNA)
may become an important therapeutic tool
is RNA interference always synthetic or created by humans?
no, it occurs naturally in earthworms, fruit flies, some fish, mice and plants
to protect them from viruses or other DNA sequences like transposons
What is dicer?
an RNase enzyme (digests RNA into nucleotides)
what is RISC
RNA induced Silencing Complex
so again, how do dsRNAs work?
inhibits protein synthesis
and
expression of complementary nucleic acid sequences
Exact mechanism of interference using dsRNA explained
1) we start with dsRNA (based on what sequence you want to inhibit
2) dicer breaks it down to siRNA (small interference RNA)
3)siRNA is unwinded from double strand to single strand using RISC
4) unwinded siRNA will bind to the sequence of RNA that we are targeting and cleave it causing it to be silenced
so again, what is siRNA?
small interfering RNA sequences
why are siRNAs beneficial?
they reduce the presence of unwanted or faulty proteins in humans and animals
how can siRNAs used for RNAi (RNA inhibition) be made?
They can either be made from
dsRNA that is injected into cells
(like we explained before)
or
it can be
endogenously encoded
(naturally)
or
from
microRNAs (miRNA) processed from stem-loop structures found within the host genome
how many miRNAs are in humans?
approximately 1000 miRNAs
what % of genes are regulated using miRNAs
30% of all genes (using many pathways)
how is miRNA made?
1) It starts as
pri-miRNA (primary) by RNA polymerase II
2) it then gets processed in the nucleus using RNAse III in vertebrates
3) RNAse III converts it into
shorter stem-loop structures dsRNA (aka: precursor RNAs and Pre-miRNAs)
4) The Shorter stem-loop structures dsRNA (aka: precursor RNAs or Pre-miRNAs) * is processed by dicer (RNAse III) into mature miRNA
how is miRNA made, simplified.
Pri-miRNA (primary) is made by RNA polymerase II
I
I
V
Processed in nucleus by RNAse III (Drosha)
I
I
V
converted into shorter stem-loop structures dsRNA
(aka: precursor RNAs or Pre-miRNAs)
I
I
V
Processed by RNAse III (dicer)
again
I
I
V
mature miRNA
What does miRNA do?
it enters RISC to target complementary mrna for degradation or translational repression
what does miRNA share pathways with?
what is the end result?
dsRNA,
it also uses RISC and is processed into siRNAs
in the end , it also exhibits a perfect sequence that is complementary (binds) to mRNA to inhibit protein synthesis
what two kinds of RNA get converted into siRNAs that bind to mRNA to inhibit protein synthesis?
dsRNA and miRNA
what is the main difference between miRNA and dsRNA
miRNA gets converted to shRNA before it is finally processed into siRNAs
what are the applications of siRNA and shRNA
there are at least 22 different siRNA/shRNA sequences being therapeutically tested for treatment of at least 16 diseases
what are the delivery methods available for siRNA or shRNA
Ex vivo
local
systemic (active or passive)
where is local delivery usually done?
it is done to tissues that are external or locally restricted
such as
epidermal, ocular, pulmonary, colonic and pancreatic
what is siRNA/shRNA used for when injected in
1)intraocularly
2)colon
1) it down-regulates beta adrenergic receptors and inhibits production of aqueous humor to relieve
intraocular hypertension
2) ceq508 shRNA oral therapy inhibits beta-catenin to slow polyp growth in FAP
(colon cancer)
what is an application for siRNA in the lungs?
Excellair (a medication that is an siRNA) inhibits spleen tyrosine kinase and slows proinflammatory transcription factors (
to treat asthma
)
what are problems associated with Systemic Iv injection of siRNA
unmodified siRNA accumulates in the kidneys
siRNA encapsulated in liposome and nanoparticles gets trapped in the liver
therapy for those organs is relatively easy as non-targeted passive delivery
how can targeted systemic delivery be possible ?
by coating siRNA with cell-specific ligands that will cause receptor mediated uptake and slow release
what is an example of using targeted systemic delivery?>
targeting the
transferrin receptor
that is highly expressed in tumor cells.
how is Ex vivo delivery achieved?
using bacterial and viral vectors
you remove the cells, infuse them with siRNA/shRNA and then return them to the patient
what is an example of EX vivo deliver?
using recombinant GM-CSF and bifunctional shRNA to down regulate furin
this indirectly reduces Tumor growth factor beta 1 and 2
(TGF-beta 1 and 2)
outcome: cause immune cells to
attack ovarian cancer cells
what activity is usually associated with Barrett's adenocarcinoma SEG-1 cells
Telomerase activity
what is used to target telomerase and treat barrett's adenocarcinoma?
the use of dye (CY-3) labeled siRNAs
resulting in inhibition of telomerase expression and loss of telomerase activity, telomere shortening
increases levels of genes that
cause cell cycle arrest and promote apoptosis
TOPIC #3
Ligand RNAs: Aptamers and Decoys
I was just letting you know the name of the topic, proceed
What are aptamers?
Single-stranded nucleic acids that bind to molecular targets with high affinity and specificity (20-100 nucleotides)
what do aptamers do?
they bind targets such as a single strand unpaired nucleotides, duplexes, triplexes, and quadruplexes (
can bind with proteins
)
they modulate protein function similar to antibodies
how can Aptamers be modified
they can be modified to increase nuclease resistance and improve pharmacological properties
or
conjugated to cholesterol or polyethylene glycol to reduce renal filtration
what are the advantages of aptamers over antibodies
Elicit minimal immunogenicity
smaller so better penetration and transport
can be conjugated to ribozymes and aptamer-siRNA
can be synthesized in large scale
can serve as delivery vehicle for siRNAs
what is an example of an aptamer
FDA approved drug Macugen:
it targets VEGF for Age-related macular degeneration (AMD)
what are obstacles of using
aptamers
minimization of immune stimulation
prolonged duration of drug (can lead to toxicity)
efficiency and specificity for delivery
what is CRISPR
Clustered
Regularly
Interspaced
Short
Palindromic Repeats
what two things is CRISPR made of?
1) a CAS enzyme
2) short palindromic repeats that are
identical
3) Spacers in between the palindromes (these are
specific
)
what are the spacers?
they contain history of old infections so the body won't be infected again
what is the mechanism of CRISPR
1) the bacteriophage attaches to the cell
2) the CAS enzyme transcribes DNA that makes
crRNA
(crisper RNA)
3) the complex of CAS+crRNA attaches to the viral dna and breaks it apart
how does the complex of CAS+crRNA break the viral DNA apart?
The crRNA (made of palindromic repeats and spacers)
**the spacers are specific to each viral dna (a specific spacer is used for a specific viral DNA)
what happens when the CRISPS doesn't have a specific spacer for the bacteriophage?
The Cas enzyme creates class 1 CAS protein, that attaches to the viral DNA and breaks it apart.
it also takes the viral DNA and copies it into the CRISPR system as a spacer (it makes a new spacer)
what is CAS 9?
a crisper system that 2 scientists made to change DNA sequences to
1) remove unwanted genes (to treat genetic diseases)
or
2) add a gene that they want
how does CAS 9 contain
1) a tracer RNA
2) a crRNA
3) 2 nucleases that break apart the target DNA
how does CAS 9 add or remove genes?
how to remove DNA:
1) breaks the target gene apart using 2 nucleases to cleave and remove it
2) after the unwanted part is cleaved and removed, the cell repairs the part by adding a mutated DNA
to add:
1) break apart the gene the same way (using 2 nucleases)
2)we insert an RNA that we made and add it to the part that CAS-9 previously cleaved
What is gRNA?
a name given to tracer and crRNA
what are the 2 enzymes used in CAS?
helicase- unwinds DNA
nuclease- cleaves DNA
;