investigating the biodistribution of mtl-cebpa reveals ...€¦ · investigating the...

Investigating the biodistribution of MTL-CEBPA reveals delivery of small activating RNA into CD34+ cells and different types of immune cells in vivoAlbert Kwok1, Nina Raulf1, Vikash Reebye2, Robert Habib1, Matt Catley1, David Blakey1, John Rossi3 and Nagy Habib2

1MiNA Therapeutics, London, UK; 2Department of Surgery, Imperial College London, UK; 3Molecular and Cellular Biology, Beckman Research Institute, City of Hope, CA, USA

Background

• MTL-CEBPA is currently in Phase Ib clinical trials in patients withadvanced hepatocellular carcinoma (HCC) (Clinical trial information:NCT02716012).

• MTL-CEBPA comprises a small activating RNA (saRNA) encapsulatedinside a SMARTICLES® liposomal nanoparticle to activate CEBPAexpression in cells.

• Our preclinical studies have demonstrated that MTL-CEBPA modulatesthe properties of immune cells in the tumor microenvironment insyngeneic mouse tumour models, improves liver function andsuppresses tumor growth in tumour models in immunocompetentmice and rats.

• Administration of MTL-CEBPA to patients increases the CEBPA mRNAexpression in circulating white blood cells.

• However, the cell types targeted by MTL-CEBPA in the immune systemare not well defined.

Aim• The aim of the study is to extend our knowledge on the biodistribution

of MTL-CEBPA to cells of the immune system.

RNA activation mechanism

Flow cytometry gating strategy MTL-Cy3-CEBPA uptake in CD34+ cells

CEBPA-51 saRNA to upregulate

CEBPA expression

NOV340 SMARTICLES®

liposomeMTL-CEBPA

API Formulation Drug Product

Hour

Intravenous Injection (3mg/kg, MTL-Cy3-CEBPA)

Blood, bone marrow and spleen were collected and cells were isolated for flow cytometry (n=5 per group)

0 4 8 16 24

• The CEBPA saRNA was conjugated with a Cy3 fluorophore and encapsulated inside the SMARTICLES® liposome to form MTL-Cy3-CEBPA.

• MTL-Cy3-CEBPA was intravenously injected in Wister rats.

• Blood, bone marrow and spleen were harvested post-intravenous injection of MTL-Cy3-CEBPA and cells were isolated for flow cytometry analysis.

Monocytes• The monocyte populations in blood, bone marrow and spleen mediate a

significant uptake of MTL-Cy3-CEPBA.

Macrophages• The macrophage populations in blood and spleen, but not in bone

marrow mediate a significant uptake of MTL-Cy3-CEPBA.

Dendritic cells• The dendritic cell populations in blood and bone marrow mediated a

significant uptake of MTL-Cy3-CEPBA.• A small population of dendritic cells in spleen also takes up MTL-Cy3-

CEPBA.

Neutrophils• The neutrophil populations do not uptake MTL-Cy3-CEBPA.

CD34+ cells• The CD34+ cells in bone marrow but not in blood uptake MTL-Cy3-

CEBPA.

T cells• The T cells in blood, bone marrow and spleen do not uptake MTL-Cy3-

CEBPA.

B cells• The B cells in blood, bone marrow and spleen do not uptake MTL-Cy3-

CEBPA.

Conclusion• MTL-Cy3-CEBPA can be taken up by some myeloid cell and

CD34+ cell populations.

Data are expressed as % of Cy3+ monocytes (CD45+ CD3- CD4+ cells). 1 way ANOVA was performed. *= p<0.05;**=p<0.01; ***=p<0.001; ****=p<0.0001 when compared to 0h.

Data are expressed as % of Cy3+ macrophages (CD45+ CD11b/c+ HIS36+ cells). 1 way ANOVA was performed. *= p<0.05;**=p<0.01; ***=p<0.001; ****=p<0.0001 when compared to 0h.

Data are expressed as % of Cy3+ dendritic cells (CD45+ CD11b/c+ OX62+ cells). 1 way ANOVA was performed. *= p<0.05;**=p<0.01; ***=p<0.001; ****=p<0.0001 when compared to 0h.

Data are expressed as % of Cy3+ neutrophils (CD45+ CD11b/c+ RP1+ cells). 1 way ANOVA was performed. No significant difference is observed between groups.

Data are expressed as % of Cy3+ CD34+ cells. 1 way ANOVA was performed. *= p<0.05;**=p<0.01; ***=p<0.001; ****=p<0.0001 when compared to 0h.

Data are expressed as % of Cy3+ T cells (CD45+ CD3+ cells). 1 way ANOVA was performed. No significant difference is observed between groups.

Data are expressed as % of Cy3+ B cells (CD45+ CD45R+ cells). 1 way ANOVA was performed. No significant difference is observed between groups.

Blood Bone Marrow Spleen

Monocytes +++ ++ +++Macrophages ++ - +++Dendritic cells ++ ++++ +

Neutrophils - - -CD34+ cells - ++ ND

T cells - - -B cells - - -

Summary of MTL-Cy3-CEBPA uptake in the cell populations 4 hour post intravenous injection. ++++ denotes ~40% of Cy3 uptake; +++ denotes ~30% of Cy3 uptake; ++ denotes ~20% of Cy3 uptake; + denotes between 5-10% of Cy3 uptake; - denotes no Cy3 uptake; ND refers to not determined.

Experimental setup

MTL-Cy3-CEBPA uptake in myeloid cells

MTL-Cy3-CEBPA uptake in lymphoid cells

Conclusion and future work

References

Future work• To investigate the CEBPA mRNA gene expression level in the

cells which uptake MTL-Cy3-CEBPA.• To apply our technology to target other diseases which can

be treated by modulating myeloid and CD34+ cells.

Loading of saRNAs into Ago2 protein

saRNA-Ago complex enters nucleus and associates with promoter region of target gene

Generation of a transcriptional activation complex to generate new mRNA

Synthesis of new protein

3.

4.

2.

1.

• Voutila J et al., Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer, Molecular Therapy, 2017

• Portnoy V et al., saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription. Cell Research, 2016

• Reebye V et al., Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo. Hepatology, 2014

• Reebye V et al., Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer. Oncogene, 2018

• Kwok A et al., Developing small activating RNA as a therapeutic: current challenges and promises. Therapeutic delivery, 2019

MTL-CEBPA Drug Product

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Viability

Live

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CD11 b/c

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OX62 Dendritic

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RP1 Neutrophils

CD11 b/c

FSC-

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CD34

investigating the biodistribution of mtl-cebpa reveals ...€¦ · investigating the...

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