1. Bioreducible cross-linked polymer coated mesoporous silica nanoparticles for targeted delivery of siRNA to HER2+ breast cancer Nanotek & Expo, San Francisco, CA December 2, 2014 Moataz Reda Biomedical Engineering (BME), Oregon Heath & Science University

2. siRNA (Small interfering RNA) - First reported in 1998, leading to the 2006 Nobel Prize for Andrew Fire/Craig Mello - Used extensively for knocking down genes of cells in vitro - Since genes/proteins are responsible for metastasis, angiogenesis, anti-apoptosis, and drug-resistance, siRNA holds great promise for cancer therapy - >85% of cancer genes are not druggable by small molecule inhibitors or antibodies. - siRNA can knock-down target genes in very specific manner 1. siRNA incorporates into RISC 2. RISC uses this siRNA strand as a template to recognize target mRNA 3. RISC then cleaves mRNA with perfect complementarity to the guide strand 4. Stop the production of that specific gene/protein (e.g., HER2). History: 2004: First human trial for local siRNA injection to treat eye disease (Age-related macular degeneration); 2009: First human trial for treating cancer using siRNA-NPs (Davis et al, Cal Tech) Abcam

3. Our target: HER2 in HER2 + breast cancer - HER2 is overexpressed in 20% of all breast cancer but treatment cost ~ 50% of all BC market - HER2 + BCs have initial or acquired resistance to drugs within a year - HER2 + BC which acquired resistance to Tykerb and Herceptin still respond to siRNA against HER2 (‘siHER2’)  siHER2 can serve as a great therapeutic if the delivery barrier can be overcome BT474-TR developed from long term Treating BT474 cells with Tykerb  Cells become resistant to both Herceptin and Tykerb However, the resistant BT474-TR cells responded to siHER2-NPs in the same manner with parental BT474

4. Cationic polymer (PEI-PEG) coated mesoporous silica nanoparticles (MSNP) conjugated with antibody for targeted delivery Core size (TEM) = 50 nm Size in water = 100 nm Our nanoparticle is designed to address the delivery barriers of siRNA (e.g., short half-life due to kidney clearance, poor cellular uptake, and siRNA degradation by blood enzyme).

5. siRNA protection from blood enzymes - Two versions of NPs (modified with 1.8kDa and 10kDa PEI) had been developed - NP with 10KDa PEI was found to be optimal; it could protect 100% of siRNA for at least 24hrs in 50% human serum - Naked siRNA had half-life of only 1 hr.

6. Nanoparticles conjugated with HER2-antibody impart delivery specificity to HER2 + cells and not HER2 - cells >90% of HER2 + cells, (SKBR3), were internalized with NP- HER2-Ab in 2 hrs, but only ~5% with NP-CD20-Ab (negative control antibody) HER2 - cells (MCF7) did not uptake the NP (<10%) Nanoparticles siRNA Nuclei Flow cytometry of dye-tagged siRNA-NP-antibody inside 10,000 cells

7. In vitro Efficacy: NP can knock down > 80% of HER2, leading to apoptotic death of HER2 + cells and not HER2 - cells > 80% HER2 knockdown vs. siX Death is specific to HER2 + cells, and not HER2 - or normal organ cells All with 60 nM of siRNA

8. Yantasee, confidential In vivo Efficacy: siHER2-NP-H knocks down 60% of HER2 in HCC1954 tumors siHER2-NP-H could inhibit growth of drug-resistant HCC1954 tumors ( 1.25 mg siRNA/kg, i.v. once a week for 5 weeks ) HCC1954 tumors grown in MFP of mice to 200 mm 3 before treatment siHER2-NP conjugated with HER2-antibody (H)

9. 9 siRNA-NP-antibody has very favorable safety profile High treatment specificity: previous slide. Blood safety (in vitro): - No hemolysis vs. PBS control - No platelet aggregation - Normal blood coagulation time Immune responses (in vitro): -Not trigger cytokine (IL-6, IL-1 , IFN- , TNF-  ) production of in blood immune cells (PBMC) compared to abraxane and feraheme

10. Acknowledgement Dr. Wassana Yantasee’s Lab –Worapol Ngamcherdtrakul –Jingga Morry –Shenda Gu –Thanapon Sangvanich –David Castro –Samuel Mihelic –Brandon Beckman –Xinran Li Joe Gray (cancer genomics) Zhi Hu (HER2-siRNA) Rosie Sears (breast cancer model)  Sponsors ◦ Prospect Creek foundation ◦ R01 from NIGMS ◦ Fast track SBIR contract from NCI (PDX Pharmaceuticals, LLC) ◦ VPR Nanomedicine Startup