Packaging of endogenous and exogenous milk miRNAs in the ...

Packaging of endogenous and exogenous milk miRNAs in the mammary gland: new opportunities for milk as a therapeutic

CSIRO HEALTH AND BIOSECURITY

Dr. Meagan Gillespie

Presentation overview

Milk miRNA biotransfer | Meagan Gillespie

miRNAs- Overview of biogenesis, distribution and packaging- Extracellular miRNAs in biofluids

Milk miRNA biotransfer- packaging of miRNAs in milk by the mammary gland- bioactivity of exogenous milk miRNAs- milk miRNAs as a therapeutic

microRNA biogenesis

milk miRNA biotransfer | Meagan Gillespie

miRNAs are

- Transcribed and cleaved to precursors in the nucleus

- Exported to the cytoplasm where they are processed, target mRNA

Joshi et al., Pulm Circ. 2011;1:357-64

microRNA tissue distribution


Liang et al., BMC Genomics. 2007; 8:166

Reproductive/glandular


miRNA biofluid distribution (extracellular)

Weber et al., Clin Chem & Genetics, 2010 Nov;56(11)


Milk-specific miRNAs (bovine)

Chen et al. Cell Res, 2010;20,1128-37

Different miRNA species in colostrum compared to mature milk

Packaging of extracellular miRNAs

Palazzo et al., Anticancer Res. 2012 Mar;32(3)

Exosomes


Ago2- Extracellular miRNAs relatively resistant to pH and temp changes

Arroyo et al., PNAS. 2011 Mar;108(12)

Mittelbrunn et al., Nat Comm. 2011 Apr vol 2


miRNA biotransfer – a biofluid messenger

- exosomes can transport biologically active miRNAs from one cell to another and between cell types

- cross-species miRNA biotransfer has been demonstrated, eg.

- honey bee and Varroa destructor parasite (Garbian et al, PLoSPathogens, 2013

- nematodes to mammalian cells and mice (Buck et al, Nat Comms, 2014)

- rice, honeysuckle miRNAs to mammals (Zhang et. al., Cell Res, 2012), refuted by Snow, Dickinson and others)


Milk miRNA biotransfer

- Baier et. al. (J Nutr, 2014) show increase in plasma miRNAs after milk ingestion, however, as these are endogenous miRNAs, the ingestion of milk may influence their expression

- Lasser et. al. demonstrated that breast milk exosomes can be taken up by macrophages (J Transl Med, 2011)

- Immunomodulatory function?


Milk as a miRNA delivery system?- Milk, the sole food source at birth, contains an abundance of miRNAs

- High proportion of these miRNAs are immune related

Kosaka et al. Silence, 2010;1(7)

- delivery to the young and modulation of the immune system?- artefact of the mammary gland?


miRNA packaging in vitro- The majority of miRNAs are non-selectively secreted into exosomes, small number are preferentially secreted

- Transfection alters miRNA packaging

Tosar et al. Nucleic Acids Research, 2015;43(11)

MEC extracellular fractions


Milk miRNA packaging in vivo- Transgenic mouse and cow developed by our collaborators (Jabedet. al., PNAS, 2012: 109(42))

- Transgene contains 2 artificial miRNAs behind WAP promoter that target B-lactoglobulin

- Founder mice have 2 inserts, both not in miRNA coding regions

- We used this mouse to examine exogenous miRNA packaging and the biotransfer of milk miRNAs in vivo


Transgenic mice and cow express high levels of exogenous miRNA in milk

Mouse 1

Mouse 2Mouse 4


Alteration of the milk miRNAome by expression of an exogenous miRNA

transgenic wild type transgenic no amiR

amiR4

Total milk miRNA population day 3

71 miRNA species expressed in tgx and wt milk; 6 differentially expressed

- amiR behind WAP promoter, random transgene integration

- Also alters mammary miRNAome; 22 de of 339


Packaging of miRNAs in milk- All miRNA species expressed in milk are expressed in mammary

gland

- amir4 is found in exosomes in mice and cow

Exogenous miRNAs are bioactive in mouse and cow


Jabed et. al., PNAS, 2012: 109(42)


Biotransfer of amiR4 in mice: Potential for delivery of miRNAs via milk

amiR4 expression in pup spleens

Lactating tgx dam

Wt pups

- Optimised “high-throughput” exosome isolation method for milk

- Currently comparing the affect of different milk processing methods on miRNA bioactivity

- Previous studies looking at miRNAconcentration found miRNA was reduced after pasteurisation(Howard et. al., J Agric Food Chem, 2015)

Implications of milk processing on miRNAbioactivity


Milk miRNA expression can be altered

- miR6-4 cow and mice express high levels of amiR4

- amiR4 is bioactive

- precision genome engineering to develop mammals expressing therapeutic small RNAs in milk

Transfer of milk miRNAs occurs in mammals

- dose-response in humans ingesting cows milk- transfer of exogenous miRNA to ingesting pup spleens

Milk processing destroys milk miRNAs

- potential to retain bioactivity of miRNAs in milk products such as infant formula by optimising processing

Summary


The biological function of milk miRNAs as an active nutritional component

Retaining the function of milk miRNAs in infant formula and other dairy products during processing

Future directions


AgResearch

Stefan Wagner

Goetz Laible

CSIRO Animal Health

Mark Tizard

Tim Doran

Tamsyn Crowley

Samantha Grimley

Susanne Wilson

Terry Wise

Kirsten Morris

Acknowledgements


CSIRO Food Sciences

Jared Raynes

Mary Ann Augustin

Packaging of endogenous and exogenous milk miRNAs in the ...

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