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Platelets are small, enucleate, multifunctional cells that modulate many important
physiological roles including hemostasis and immune signalling. Currently !"#$%&'(')*+,*-%.#')%#)%'/01*#-)'2.3%,-%/#'2/#%45)%..#678
transfused as a therapy to maintain hemostatic balance, platelets are an integral
A
part of hemorrhage management. However, transfusions can be ineffective in the A. Confocal microscopy images of

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most severe cases of hemorrhage. In addition to hemostasis, platelets are a megakaryocytes treated with 1.0 !g/mL LNP (top)
supplemented with 1.0 µg/mL ApoE and labeled
potential cell therapy in other applications, but development has been hindered with the megakaryocyte-specific cell markers,
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by inadequate methods to control which proteins are expressed by platelets. For CD41a and CD61, demonstrate GFP expression
example, there are no methods to express exogenous proteins in transfusable and successful transfection, as compared with
the untreated cells (bottom).

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platelets, which would expand their use to help treat the diseases they modulate.
Current methods to modify platelets largely entail viral transduction of 50 µm

megakaryocytes (MKs), whereby proteomic changes in the MK are inherited by

the resulting platelets. The use of viruses, however, present challenges in B–D. Nearly all megakaryocytes express GFP when treated with as low a dose of 0.3 !g/mL
efficiency and scale-up for clinical use. Another method that is easily amenable mRNA-LNPs and supplemented with 1.0 µg/mL ApoE, with increased GFP fluorescence
intensity above 1.0 !g/mL of mRNA-LNPs. Megakaryocytes were identified as those positive
to scale-up for the clinic is therefore needed to produce modified, transfusable for the CD41a and CD61. Error bars represent SEM.
platelets, and thus enhance their protein composition for specific applications.
B C D GFP B525-FITC-A-
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To develop methods needed to produce modified platelets by directly 0

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transfecting donor-derived platelets and megakaryocytes (MKs) with mRNA via !"#$"%&'()*+),+)$-,.),*/.0
Comp-FL1-A :: GFP B525-FITC-A
0.3 !g/mL

lipid nanoparticle (LNP)-mediated delivery. LNPs have already demonstrated Key Takeaways
clinical safety and efficacy for gene therapy1,2, and cultured MKs provide an ! Megakaryocytes from cord-blood HSCs are easily transfected in vitro with mRNA-LNPs with
alternative source of cells that can be engineered to produce modified platelets3. high efficiency
It is therefore hypothesized that, as an initial proof-of-concept, LNPs containing ! mRNA-LNP dose of 1.0 µg/mL appears optimal for high transfection efficiency and protein
mRNA encoding for GFP can be delivered to MKs and platelets to engender GFP production
! Next step: Verify that platelets produced also inherit changes and express physiologically-
expression in both cell types, which can ultimately be extended towards other
relevant protein
proteins to enhance the natural coagulability and functional repertoire of
platelets.

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! Lipid nanoparticles containing mRNA (mRNA-LNPs) encoding for GFP were

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3 3 3 1.0K
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600
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formulated and used to treat both megakaryocytes and platelets at varying 0 0 0
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mRNA-LNP doses.
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300
0.3 !g/mL 1.0 !g/mL
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5
10 0,032 99,610 0,045 99,5
Comp-APC-A Comp-APC-A Comp-APC-A

10
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oFor megakaryocytes: The MKs, derived from cord-blood hematopoietic stem cells (HSCs),
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were treated with the mRNA-LNPs with or without 1 µg/mL of apolipoprotein E (ApoE) to
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facilitate LNP uptake. 12345

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oFor platelets: Donor platelets were washed and treated with the mRNA-LNPs but DiI-labeled mRNA-LNPs, with 1.5K
900
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increasing DiI intensity in a dose-
Count

incorporating the lipophilic tracer, DiI, to track particle uptake alongside expression. dependent manner suggesting
1.0K
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increased LNP uptake. Error bars 500

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mRNA-LNPs Dose mRNA- mRNA-LNPs Dose mRNA-

represent SEM.
0
LNP (µg/mL) LNP (µg/mL) 10
1
10
2
10
3
10
4
10
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Comp-FITC-A
! 0.3 ! 0.03
! 1.0 ! 0.1
! 1.5 ! 0.3
! 2.0 DiI-labeled ! 1.0
Media D-E. At the highest mRNA-LNP dose of 1.0 µg/mL and 24 hours post-treatment,
!Serum-free Donor
!+1.0 µL/mL ApoE Platelets approximately 30% of platelets express GFP after 24 hours, demonstrating the ability of
platelets to take up and express exogenous mRNA. Error bars represent SEM.
24 hrs
Washed platelets
Panel
Panel 24 hrs !GFP Key Takeaways
!GFP
!CD41a-PE
!DiI (PE)
!CD42b-APC
! Washed donor platelets easily take up mRNA-LNPs even at very low doses
!CD61-APC ! Higher mRNA-LNPs doses can lead to expression of exogenous mRNA
! Next steps: Optimize LNPs for higher mRNA expression levels, and express physiologically-
relevant proteins
Flow cytometry Confocal Microscopy
Flow cytometry

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! Megakaryocytes and platelets can readily be transfected with LNPs to produce exogenous protein.
! The production of modified platelets in vitro will need to be verified, with resulting platelets extensively characterized to determine changes in physiologic functions.
! The difference between LNP uptake and expression of delivered mRNA in ex vivo-treated donor platelets also suggests that some of the mRNA may still be sequestered or
inaccessible to translation.
! If all is successful, the modified platelets can be transfused into animal models to determine their efficacy as a novel treatment towards various platelet-related diseases.
Alternatively, modified megakaryocytes can be transfused to produce modified platelets directly in the body.
! Eventually, this can lead to the creation of a platform technology that in the long-term will allow platelets to deliver therapeutic proteins and yield more effective platelet
products.

%!+#",-./0.*.#$1 !"#$%!$&'#(")*%$'"#&9 [email protected]

J. Leung is supported by the Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award. D. Witzigmann acknowledges support from the Jerry Leung | PhD Candidate
Swiss National Science Foundation. We would also like to thank the Canadian Blood Services and the blood donors for providing the pooled platelet products.
Department of Biochemistry and Molecular Biology
University of British Columbia, Vancouver, BC, Canada

1. Kulkarni J-A et al. Lipid nanoparticle technology for clinical translation of siRNA therapeutics. Acc Chem Res 2019; 52(9);: 2435-2444 3. Ito Y et al. Turbulence activates platelet biogenesis to enable clinical scale ex vivo production. Cell 2018; 174;: 638-648
).(.).#!.1 2. Akinc A-M et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol 2019;
14(12);: 1084-1087