1. VIROMER® Transfection Technology From iN ViTRO to iN ViVO - mRNA delivery -

2. Immune cells are „resistant“ to plasmid transfection Macrophages Monocytes Dendritic cells …are known to be very hard-to-transfect with plasmid DNA. Why? Too high DNase activity in the cytosol! Foreign DNA  Activation of enzymatic cascades e.g. AIM2-Interleukin cGAS-Interferon Innate immunity

3. Examples of data: Plasmid transfection with VIROMER® RED is possible… Order #: VR-01LB-00, VR-01LB-01 or VR-01LB-03

4. … but it’s limited and not always reliable! Macrophages can take up DNA

5. Anonymous user’s data THP-1 monocytes: up to 25% of transfected cells (after optimization)

6. Anonymous user’s data … but it’s toxic! THP-1 monocytes: up to 25% of transfected cells (after optimization)

7. mRNA is a great alternative!

8. Data from S. Fabb, Monash Institute of Pharmaceutical Sciences, Victoria, Australia Transfection of GFP encoding mRNA in cells seeded in RPMI (10% FCS) or OptiMEM® medium (0% FCS) with Viromer® RED Start Positive® Control THP-1 monocytes: up to 50% of transfected cells

9. 90% Human Primary Monocytes: up to 90% of transfected cells

10. Human Primary Monocyte-derived Macrophages: up to 55% !!

11. …and Monocyte-derived Dendritic cells: 40-50% “[..] We used Viromer red to transfect mRNA (a GFP control RNA) into monocyte-derived DCs. The transfection efficiency was 40-50% with excellent viability (no signs of cell death).” University of Florida, 25 th July 2016

12. Make the bridge from in vitro to in vivo!

13. Like in this paper… VIROMER® RED was used in vivo! Nature (2016) doi:10.1038/nature18300 We have now developed a specific formulation for this application 

14. In vitro VIROMER® reagents: optimized to work equally strong with DNA and mRNA You can test it with the Start Positive® Controls ! Preformed VIROMER® complexed with pCMV-GFP plasmid (1 vial) and GFP encoding mRNA (1 vial) Work with mRNA also in vivo !

16. Viromer® IN VIVO = Lyophilized Viromer® + water + mRNA (=cargo) Injection of the complex into living organisms Systemic route - intravenous tail injection Low dose mRNA: spleen targeting Resident macrophages and circulating monocytes High dose mRNA: spleen + liver Kuppfer cells, endothelium, hepatocytes Mix and wait 5 min Straightforward Protocol

17. Selective delivery into spleen using VIROMER®: mRNA complex 2h 6h 44h TOP: Rapid and durable transfection of luciferase mRNA into spleen. Signal appears as early as 2h after tail vein injection, peaks at 6h and expressed protein is still active after 44h. (10µg mRNA formulated using specific Viromer®, mice seen from dorsal side) RIGHT: luminescense of isolated organs confirms exclusive delivery to spleen Lung Liver Spleen Kidney

18. Strong expression in liver using Viromer®: mRNA complex 25µg 40µg 6h 24h 24h, isolated organs Lung Liver Spleen Kidney Rapid and strong transfection of luciferase mRNA into liver. Using 25 or 40µg of mRNA  preferred liver targeting is achieved. Expression in liver is very pronounced after 6h and still detectable after 24h. Luminescense in isolated organs confirms specific expression in liver and spleen, but no other organ.

19. VIROMER® IN VIVO mRNA - Systemic Kit #Vivo-01mRNA-02 Application Intravenous injection in the tail vein Target resident macrophages/monocytes of the spleen (low dose) or the liver (high dose) Content 6 vials @ 5 injections (25-g mouse) = 30 rxns 2 vials of Positive Controls Price 1250€ https://viromer-transfection.com/product/viromer-in-vivo-mrna-systemic-kit Product Information

20. Upcoming products for local injections (September 2016) Local injections are a great way to localize the expression of gene products to a certain target site. Muscles, skin, intestine & peritoneum can easily be reached using direct injections. Local mRNA delivery in mouse skin, muscles and intestine Expression of luciferase is clearly visible as a localized signal. Luciferase encoding mRNA were complexed to Viromer and signals were imaged 6 hours upon intradermal and intramuscular 5-µg mRNA injections (dorsal view). Broad expression of luciferase was detected in peritoneum and is extended to the spleen. Mouse received 15µg of mRNA via intraperitoneal injection. Luciferase was monitored after 6h. (ventral view). Expression of luciferase was detected in duodenum and small intestine. Mouse received 15µg of mRNA via intraduodenal injection. Luciferase was monitored after 6h. For imaging purposes, the abdomen was opened and the intestine exposed. (ventral view). Intradermal Intramuscular Intraduodenal Intraperitoneal spleen