mirVana microRNA mimic and inhibitors

Transcription

The next generation of miRNA modulators: new mirVana™ miRNA mimics and inhibitors
Dr. Amy Lam
Field Application Scientist
Noncoding RNAs?? Noncoding transcripts
Regulatory ncRNAs
miRNA (microRNA)
piRNA (piwi-interacting RNA)
Housekeeping ncRNAs
snoRNAs
tRNA (transfer RNA)
rRNA (ribosomal RNA)
snRNA (small nuclear RNA)
siRNA (small interfering RNA)
tmRNA (small nuclear RNA)
rasiRNA
(repeat-associated siRNA )
RNase P RNA
eRNA (efference RNA )
gRNA (guide RNA)
pRNA (promoter RNA )
MRP RNA
(mitochondrial RNA processing RNA)
scaRNA
(small Cajal body specific RNA)
long ncRNA (Xist etc.)
vRNA (viral RNA)
SRP RNA
(signal recognition particle RNA)
Telomerase RNA
Noncoding RNAs may contribute to the complex networks that regulate cell
function and could be the answer to the genome complexity.
2
6/4/2012 | Life Technologies™ Proprietary and confidential
Mechanism of RNAi:
Natural mechanism:
siRNA: mediates degradation (knockdown) of
a target mRNA molecule in a sequencespecific manner.
miRNA: very similar, but typically suppress
translation without cleavage
1000 miRNAs, target and regulate about 50% of mammalian genes
Complicated & poorly understood
3
microRNA
miRNA Functions
• Development (cell fate)
• Cell death (apoptosis)
• Epigenetics
Human Disease
• Diagnostic markers
• Therapeutic targets
• Therapeutic agents
*mature active
strand
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Strong Links to Cancer
MicroRNAs Act Both as Oncogenes & Tumor Suppressors
vector
mir-34a
mir-34b/c
5
Tumor Suppressor: miR-34
Oncogene: miR-19-92
He (2007) Nature
He (2005) Nature
What Are My Customers Asking?
6
Life Has Small RNA Researchers Covered
Biological Which miRNAs
Question are expressed?
Applications Discovery
Technology
Where and when are
miRNAs expressed?
Which genes &
cell processes?
Validation/
Screening
Profiling
Next Gen Sequencing
Instruments
Ion Torrent PGM Sequencer
Products
Ion Total RNA-Seq Kit
Microarray
Real Time PCR
Functional
Analysis
RNAi
Real Time Instruments
(StepOnePlus™, 7300, 7500
& 7900 HT)
TaqMan® miRNA Assays
& Arrays
Custom TaqMan®
Small RNA Assays
mirVana miRNA
Mimics & Inhibitors
Lipofectamine™
RNAiMAX
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mirVana™ mimics and inhibitors- novel tools
for miRNA functional analysis
Mature miRNAs enter the
miRNA pathway when
transfected into cultured
mammalian cells
mirVana™ miRNA mimics
These are double‐stranded oligonucleotides (with 2nt overhangs) that mimic the natural microRNA. These molecules are not processed by RISC machinery, they don’t have any loops or additional bases.
Antisense oligonucleotides
bind and inhibit miRNAs
mirVana™ miRNA inhibitors
These are single‐stranded oligonucleotides
with chemical modifications, that irreversibly bind and inactivate the natural miRNA.
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mirVana™ miRNA Mimics and Inhibitors
Potency
Specificity Stability In Vivo
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mirVana™ miRNA Inhibitors
Enhanced Potency
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mirVana™ miRNA mimic mature strand is highly potent
while “star” strand is inactivated
Mature miRNA (Guide) strand
Star (Passenger) strand
miRNA
Luc
Luc
miRNA Target
Reverse Target
1.200
1.400
1.000
1.200
1.000
Fold Change
Fold Change
0.800
0.600
0.800
0.600
0.400
0.400
0.200
0.200
0.000
0.000
miRNA miRNA miRNA miRNA miRNA miRNA
mimic 1 mimic 2 mimic 3 mimic 4 mimic 5 mimic 6
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Neg
miRNA miRNA miRNA miRNA miRNA miRNA
mimic 1 mimic 2 mimic 3 mimic 4 mimic 5 mimic 6
Neg
mirVana™ Mimics:
Highly functional in reporter assays
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mirVana™ miR1 mimic:
Significant down-regulation of PTK9 mRNA target
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10
9
8
7
6
5
4
3
2
1
0
3 nM
mir19a
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miR23a
Let7c
Let7a
neg
inhibitor
neg
inhibitor
neg
inhibitor
neg
inhibitor
neg
0.3 nM
inhibitor
Fold change
mirVana™ miRNA inhibitors display high functionality
in reporter assays
miR21
mirVana™ Inhibitors:
More potent than lead competitors
Remaining free miRNA – small RNA TaqMan assays
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Potency
Specificity
Stability In Vivo
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mirVana™ miRNA mimics
Enhanced Specificity
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mirVana™ Mimics Strand Specificity:
Active mature strand/inactivated star strand
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mirVana™ Inhibitors:
Target specific
3nM
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Potency
Specificity Stability
In Vivo
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mirVana™ mimic Stability:
Over 1 week at +37ºC
Over40 freeze/thaw cycles
Integrity was also confirmed by HPLC
Inhibitors also tested for stablity at +37C and freeze/thaw cycles
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mirVana™ miRNA inhibitors are stable for 1 year at +4ºC
8
3nM
7
0.3nM
Fold Change
6
5
4
3
2
1
0
inhibitor 1 year
inhibitor 2 months
inhibitor 1 week
Neg
Real-time stability data. miR-23a inhibitors, 3 batches: synthesized
1 yr, 2 mo and 1 wk before test. Storage conditions: 20 μM water stocks, +4ºC
•In addition to functional test, integrity of mirVana inhibitors was confirmed by HPLC
•Inhibitors were also stable upon storage for 1 mo at +37C and 40 freeze/thaw cycles
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Potency
Specificity Stability In Vivo
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New Delivery Reagent — Invivofectamine® 2.0
Lipid nanoparticle reagent
Designed for systemic liver delivery
Potent at low dose (<5mg/kg)
Easy to use
Robust
Phalloidin
siRNA
Hoechst
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Non toxic
Invivofectamine® 2.0: novel reagent for small
RNA delivery in vivo
Biodistribution
Alexa 647 labeled oligo
Cryosectioning
Dapi and Phalloidin staining
Small RNA TaqMan assays
Injection of the miRNA mimic or
miRNA inhibitor nanoparticles
24-72h
Sacrifice animals, harvest organs and blood
Analyze the biological effect
mRNA: qRT-PCR
Protein: Western or PLA
120
protein
Remaining (%)
100
mRNA
Toxicity
Liver tox panel
Cytokines
Hematology
80
60
40
20
0
FVII Ambion siRNA
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Neg siRNA
untreated
mirVana™ miRNA mimics are efficiently delivered to the liver
with Invivofectamine® 2.0 in the biologically active form
120
PTK9 mRNA remaining (%)
100
80
60
40
20
0
miR1 mimic
siNeg
NT
Down-regulation of PTK9 mRNA following in vivo delivery of the
miR1 mimic/Invivofectamine 2.0 at 7 mg/kg dose
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mirVana™ miRNA inhibitors are efficiently delivered to the liver
with Invivofectamine® 2.0 and suppress microRNA targets
Upregulation of four miR122 target genes – following miR122 inhibition (murine liver).
Daily injections (3 days) of inhibitor/Invivofectamine 2.0 (7mg/kg )
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mirVana™ Mimics & Inhibitors:
Non-toxic in vivo!
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Custom: Mimics, Inhibitors, & siRNAs can be fluorescently
Labeled w/o affecting activity
siRNA GAP (3000ms)
siRNA ALDH1B1 (3000ms)
HeLa cells transfected with siRNA-Alexa 647-> 24hrs later cells washed->
fixed with 4% formaldehyde-> washed 2x-> DAPI mount
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Custom option: miRNA mimics, inhibitors or siRNAs can be fluorescently
labeled to allow visualization of uptake w/o affecting activity
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Mouse #1: RNA - Alexa 647
Mouse #2: RNA - Alexa 647
Mouse #3: unlabeled RNA
Mouse #4: untreated
Alexa 647-labeled small RNAs are readily visualized in the liver sections, following systemic
delivery with Invivofectamine 2.0. Green-Phalloidin, Blue-Dapi,
Red-Alexa
647
6/4/2012
| Life Technologies™ Proprietary and confidential
Summary:
9Potency
9Specificity 9Stability 9In Vivo
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What makes mirVana™ mimics & inhibitors better?
¾ Inhibitors: much more potent due to novel chemical modifications
(specificity is about the same, impossible to enhance w/o sacrificing potency)
¾ Mimics: more specific due to chemical modifications inactivating star strand
(potency is about the same, impossible to boost it as we're mimicking
the natural miRNA)
¾ Both mimics and inhibitors are now available at in vivo scales & purity
¾Latest Sanger version V16.0 – for tubes and libraries
¾ Standard purity: desalted (instead of HPLC) - better turn around time
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The complete solution for in vitro & in vivo
miRNA functional analysis
RNAiMax® reagent (in vitro)
Invivofectamine® 2.0 reagent (in vivo)
mirVana™ miRNA inhibitors (in vitro, in vivo)
mirVana™ miRNA mimics (in vitro, in vivo)
•In vitro: 5 and 20 nmols, standard purity, desalted
•In vivo: 250 nmols, HPLC purified, in vivo ready (IVR)
•Human, mouse, rat Libraries
•Latest content of miRbase
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Which miRNA Mimic & Inhibitor is Right? Value Continuum
Performance
Performance Plus
Standard
Performance Plus
Product
Brand
Ambion® PremiR Precursors
mirVana™
miRNA Mimics
Ambion® AntimiR Inhibitors
mirVana™
miRNA Inhibitors
Technology
Proprietary
(2nd Generation)
Proprietary
(3rd Generation)
Proprietary
(2nd Generation)
Proprietary
(3rd Generation)
Attribute
Novel design to minimize off‐
target effects
Next generation chemistries for lowest off‐target effects
Chemically modified for good efficacy
Next generation chemistries for highest efficacy
Content Database
100% Coverage of
miRBase v 15*
100% Coverage
of miRBase v 17*
Function
Species
Mimic endogenous miRNAs
Human
Human, Mouse &
Rat
(19 other species
as customs)
100% Coverage 100% Coverage of
miRBase v 17*
of miRBase v 15*
Inhibit endogenous miRNAs
Human
Human, Mouse &
Rat
(19 other species
as customs)
* mirBase : v15 - 1090 distinct human sequences
v17 - 1719 distinct human sequences
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mirVana microRNA mimic and inhibitors

Transcription

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