Liposomes for micro-RNA delivery - Nano2Clinic · Liposomes for micro-RNA delivery 1. ... Confer high stability to miRNA molecules ... Translational Aspects ..... 1st Food and Drug

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: ruth.prassl@medunigraz.at

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

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

+

-

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

)

0.0 0.1 0.2 0.3 0.4 0.5

I (a

rb. u

nits)

0

2000

4000

6000

8000

10000

z (Å)

-40 -30 -20 -10 0 10 20 30 40

(

arb

. u

nits)

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

DSC

SAXS

TEM Analytics

• Biological Assays

• Electrophoretic Techniques

• RT-qPCR

Adipositas - Brown Fat in Humans

-uptake

energy uptake = energy expenditureBC + EXbasal exercise

consumption

+

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)

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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+

++

+

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Toxicity : MTX and LDH

Screening of Lipid Formulation and miRNA/liposome complexes

Nat

ive

PA

GE

miRNA + + + + +liposome - + + + +

triton - - + + -nuclease - - - + +

miRNA-26a (22nt)

successful liposomeloading

effective microRNAprotection

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

rela

tive

UC

P1

leve

ls

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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