Glucocorticoids modulate micro rna expression and processing during lymphocyte apoptosis
Liposomes for micro-RNA delivery - Nano2Clinic · Liposomes for micro-RNA delivery 1. ... Confer...
Transcript of Liposomes for micro-RNA delivery - Nano2Clinic · Liposomes for micro-RNA delivery 1. ... Confer...
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nanomedicine
Ruth Prassl
Gottfried Schatz Research CenterCell Signaling, Metabolism and Aging
Drug Delivery & Imaging
Contact:
Dr. Ruth Prassl
Medical University of GrazGSFZ - BiophysicsNanomedicine GroupNeue Stiftingtalstraße 6A-8010 Graz
Phone: +43 (0)316 385-71695E-Mail: [email protected]
Liposomes for micro-RNA delivery
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cancer
• miRNA are non-coding RNAs
• miRNA target multiple mRNAs
micro RNAs – miRNA therapeutics
RNA-induced silencing protein complex
miRNA in RISC interacts with mRNA
gene expression gene expression
19-25 nucleotides in length
mimicendogenous miRNA
inhibitendogenous miRNA
miRNA therapeutics
Modulation of disease pathways2
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Lipid based miRNA delivery systems
Need of a delivery system
High encapsulation efficiency and long-term stability
Protection of miRNA from degradation
Confer high stability to miRNA molecules
Improvement of pharmacological properties
Opportunities for tissue specific targeting
Low immunogenicity and toxicity
Safe delivery to diseased site with reduced risk of off-target effects
Further Requirements
Robust and reliable production processes
Scalable and cost efficient
Cationic liposomes
(Cullis and Hope, 2017)
Lipid NPs with ionizable cationic lipids
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Lipids/formulations :
DOTMA/ DOTAP/ DC-Chol as cationic lipid
DOPE as neutral lipid („helper lipid“)
POPC
± Hyaluronic acid
Stearylamine / Protamine
Coating with PEG or PVP
Incubation15 min RT withmiRNA
Cationic liposomes
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Lipsomes/miRNA complex
Transfection
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Liposomes for microRNA delivery
Liposome characterisation :
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Composition
Size
Charge
Loading
Stability
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Liposome Particle Manufacturing Techniques
• Lipid Film Rehydration Technique followed by Size Extrusion or Sonication
• Ethanol Injection Technique
• Microfluidics
... easy scale-up for large preclinical studiesGMP production Dialysis
Ultrafiltration
Additional steps
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Biophysical Techniques
• Photon Correlation Spectroscopy
• Nanoparticle Tracking Analysis
• TEM /SEM / AFM
• Zetapotential Measurements
• Small Angle X-Ray Scattering
• Differential Scanning Calorimetry
Physicochemical Characterisation Techniques
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q (Å-1
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0.0 0.1 0.2 0.3 0.4 0.5
I (a
rb. u
nits)
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z (Å)
-40 -30 -20 -10 0 10 20 30 40
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arb
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nits)
-1.5
-1.0
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1.0
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DSC
SAXS
TEM Analytics
• Biological Assays
• Electrophoretic Techniques
• RT-qPCR
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Adipositas - Brown Fat in Humans
-uptake
energy uptake = energy expenditureBC + EXbasal exercise
consumption
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expenditure
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Energy balance
weight maintenance
+thermogenesis
heat
+ Tthermo-genesis
Active brown adipose tissue exists in adult humans!J. Nedergaard, T. Bengtsson, B. Cannon, Am. J. Physiol. Endocrinol. Metab 293, E444-452 (2007) K. A. Virtanen et al., N. Engl. J. Med 360, 1518-1525 (2009); W. D. van Marken Lichtenbelt et al., N. Engl. J. Med 360, 1500-1508 (2009) ;A. M. Cypess et al., N. Engl. J. Med 360, 1509-1517 (2009)
brite/brownadipocyte
(BAT)
whiteadipocyte
Energy storage
Excess Energy
(WAT)
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whiteadipocyte
briteadipocyte
MicroRNA
to convertadipocytes
adipocyteprogenitor
MicroRNA-26 Family
shifts human adipogenesisto brown characteristics
Karbiener et al., Stem Cells (2013), Karbiener et al. Meth.Mol.Bio. (2015)8
Turning fat-storing white adipocytes into fat-burning adipocytes (brown-like, brite or beige)
Strategy to fight overweight and obesity
• small multilocular lipid droplets
• high density of mitochondria
• positive for UCP1 protein in mitochondria
• massive energy dissipation
via thermogenesis
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• Size: 100 - 130 nm
• Zeta potential: liposome 44 ± 10 mV
liposome/miRNA complexes 42 ± 10 mV
Functional Unit
Neutral Lipid DOPE
Cationic LipidDOTAP
(DC-Cholesterol)
StabilizingPolymer
DSPE-PEG2000
% molar ratio 45/49/6
Lipid concentration(mg/ml)
3.0-7.0
miRNA (nM) 11-22
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Toxicity : MTX and LDH
Screening of Lipid Formulation and miRNA/liposome complexes
Nat
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PA
GE
miRNA + + + + +liposome - + + + +
triton - - + + -nuclease - - - + +
miRNA-26a (22nt)
successful liposomeloading
effective microRNAprotection
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Transfection of adipocytes – “Browning“
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human Multipotent Adipocyte Derived stem (hMADs) cells
Readouts: Elevated levels of UCP1
• Enhanced levels of miR-26a• Repressed target level of ADAM17• Long-term activation of PPARγ• Increased energy expenditure in terms of oxygen consumption
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rela
tive
UC
P1
leve
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white adipocyte
brite / beigeadipocyte
UCP1+ adipocyteUCP1- adipocyte
HPF: HiPerfect Transfection ReagentmiR-NTC : non targeting control
White adipocytes
Brown/brite adipocytes
UCP1 Immunostaining
Best Formulation …..
Transfection of adipocytes – “Browning“
RT-qPCR results
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Next Steps .....
Route of Application
subcutaneous implantation
microperfusion pumpAnimal models
Peptides
Aptamers
Active targeting of adipocytes
Alzet® Minipump
• Evaluation of in vivo activity of miR-26a• Evaluation of the composition of adipose tissues• Evaluation of the metabolic parameter
(body weight, lean blood glucose, insulin resistance etc)
Toxicity & Immunogenicity
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Translational Aspects .....
1st Food and Drug Administration (FDA) approved RNAi therapeutic in 2018Liposomal Patisiran (marketed as Onpattro® by Alnylam Pharmaceuticals) for the treatment of hereditary transthyretin-mediated (ATTR) amyloidosis.
MRX34 (MiRNA Therapeutics) a liposomal miR-34 mimic was the first miRNA therapeutic that has entered a phase 1 clinical trial in 2013 in patients with advanced solid tumors. Halted in 2016 following immune-related adverse effects.
Miravirsen (Roche/Santaris) a liposomal antagomir targeting miR-122 to treat hepatitis C is in phase 2
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Health Improvement
Treating cancer, obesity, related metabolic disorders…
Conclusion
Liposomes are a versatile and flexible nanoparticle platform
Used as universal delivery system for nucleic acids
Make your formulation as simple as possible
microRNA liposomes
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Acknowledgements
Gebhard SchratterGunter AlmerCaroline Vonach
Marcel Scheideler
Michael Karbiener
Amri Zoubir
Université de Nice Sophia-Antipolis