Evaluation of RNA interference (RNAi) in Skin

Transcription

1 Evaluation of RNA interference (RNAi) in Skin Next Generation in RNAi Pamela A. Pavco VP Pharmaceutical Development 3rd International SCAR Club Meeting March 26, Massachusetts, USA

2 2 Forward Looking Statement This presentation contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of Such statements include, but are not limited to, statements about the future development of RXi Pharmaceutical Corporation s products. These forward-looking statements about future expectations, plans and prospects of the development of RXi Pharmaceutical Corporation s products involve significant risks, uncertainties and assumptions. Actual results may differ materially from those RXi Pharmaceuticals Corporation contemplated by these forward-looking statements as a result of the risk factors discussed in RXi reports on file with the U.S. securities and exchange commission including, but not limited to, the Registration Statement on form S-1 filed with the SEC. RXi Pharmaceuticals Corporation does not undertake to update any of these forward-looking statements to reflect a change in its views or events or circumstances that occur after the date of this disclosure.

3 3 RXi Pharmaceuticals: Corporate Overview Discovery-stage RNAi therapeutics company Co-founded by 2006 RNAi Nobel Prize Recipient, Dr. Craig Mello Began operations Q1 07 Became a public company March 08 NASDAQ (RXII) Based in Worcester, MA, w/ a business office in Cambridge, MA

4 4 Basic Mechanism of RNA Interference RISC driven binding of sirna to target Target RNA is cut and destroyed, blocking deleterious protein expression Image from Robinson R (2004) PLoS Biol 2(1): e28

5 5 RNAi as a Therapeutic: Advantages Rational drug design for any gene target for which sequence is known Rapid identification of selective, potent drug candidates Can target any protein, as well as previously non-druggable targets (e.g., regulatory RNAs) Uses an efficient cellular process already present in every cell Very potent mechanism Catalytic ONE RNAi complex can target and destroy many mrnas Degraded mrna cannot be translated into protein For every mrna degraded by the RNAi mechanism, 100s to 1000s of copies of the protein are NOT made Once loaded in RISC, may have an extended duration of effect Can target multiple genes in one therapeutic

6 6 RNAi as a Therapeutic: Hurdles Compound-related Stability Potency Specificity Immune Stimulation Delivery-related RISC driven binding of sirna to target Target RNA is cut and destroyed, blocking deleterious protein expression Image from Robinson R (2004) PLoS Biol 2(1): e28 Distribution TO the tissues of interest Delivery INTO the cells of interest

7 7 Self-delivering RNAi Compounds Specific and proprietary configuration of chemistry modifications and duplex size Termini chemical moieties sd-rxrna TM Internal chemical modifications Small duplex (<15 bp)

8 8 Broad Potential Therapeutic Applications Local delivery Multiple target tissues: - compromised skin - inflammation sites - mucosal - lung - CNS (pump) - eye Systemic delivery Multiple target tissues: - liver - lung - adipocytes - cardiomyocytes - bone marrow - inflammation sites - tumors - vascular endothelium - kidney

9 Self-Delivering rxrna (sd-rxrna) Has Improved Drug-Like Properties 9 Serum stability Reduced renal clearance Target tissue uptake and penetration Minimal immune response activation Spontaneous cellular uptake Productive RISC loading Spontaneous Uptake in Cultured HeLa Cells sd-rxrna (DY547) Red Nuclei (Hoechst) Blue

10 Efficient Cellular of sd-rxrna TM Uptake Within Minutes Salomon W (2009) RXi unpublished Serum stability Reduced renal clearance sd-rxrna Target tissue uptake and penetration Minimal immune response activation Spontaneous cellular uptake Productive RISC loading Cholesterol conjugate sirna* <5 minutes incubation before wash; Cells fixed immediately RNAi compound (DY547) - Red Nuclei (Hoechst) - Blue *Described in Soutschek et al. (2004) Nature 10

11 Efficient Uptake of sd-rxrna TM into Diverse Cell Types Serum stability SH-SY5Y (neuroblastoma derived) Primary mouse macrophages Reduced renal clearance Target tissue uptake and penetration Minimal immune response activation Spontaneous cellular uptake Productive RISC loading RNAi compound (DY547) - Red Nuclei (Hoechst) - Blue ARPE-19 (retinal pigment epithelium) Primary mouse hepatocytes 11

12 sd-rxrna Directs Significant Reduction of Targeted mrna 12 Salomon W (2009) unpublished 120 Target Expression (%) Relative to Control % 70% Passive transfection of HeLa cells for 1 or 24 hours Active sd-rxrna vs. non-targeting duplex control Targeted mrna quantified at 48 hours using a bdna assay 0 sd-rxrna Control sd-rxrna Control 1 hour exposure 24 hour exposure

13 Schematic Representing Compromised Skin Model 13 Multiple incisional wounds possible on a mouse or rat 1 cm long, full depth epidermal / dermal incisions will be used to study compromised skin Treatment could be at the time of or post incision Biopsies of skin tissue harvested over time Sections (5 um thick) are paraffin embedded, formalin fixed Example section below is stained with Sirius red to show connective tissue

14 Visualization of sd-rxrna in Skin after Intradermal Administration RNAi compound (DY547) - Red Nuclei (Hoechst) - Blue Contrast Image Overlay 14

15 In vivo Silencing with sd-rxrna Following Intradermal Injection (Rat) 15 MAP4K4 Expression (% of PBS) MAP4K4 Silencing in Rat Skin Experiment 1 p = < Active vs. MM MAP4K4 sd-rxrna MM control sd-rxrna PBS % % MAP4K4 Silencing in Rat Skin Experiment 2 p = Active vs. MM MAP4K4 sd-rxrna MM control sd-rxrna PBS sd-rxrna injection sd-rxrna injection Day Incision (15 min) Biopsy harvest SD rats, n=8 incision sites per treatment, 4 sites PBS 300 ug per injection, 2 injections a day apart 6 incisions/rat, 1 cm full face incisions Two 3 mm biopsies/site processed separately RNA isolated and target expression determined by QPCR Expression normalized to housekeeping gene cyclophilin B

16 In vivo Silencing with sd-rxrna Following Intradermal Injection (Rat) 16 Single dose is as effective as two doses Presence of fluorescent label has no effect Relative Quantitation (Norm to PPIB) MAP4K4 expression in rat skin sd-rxrnas Dy547 Fluorescent Label PBS 52% p = MAP4K4 2 doses 48% p = MAP4K4 1 dose Control 2 doses Control 1 dose MAP4K4 expression in rat skin sd-rxrnas No Fluorescent Label PBS 53% p = MAP4K4 2 doses 47% p = MAP4K4 1 dose Control 2 doses Control 1 dose SD rats, intra-dermal injections; 6 small injections/dose Single administration of 300 ug in PBS on days 1 & 2 (2 doses) or on day 2 only (1 dose) MAP4K4 targeting sd-rxrna or non-targeting control, with or without Dy547 label n=5 incision sites per treatment, 1 cm incisions 3 mm skin biopsies harvested 48 hours after last dose and processed for RNA prep Target expression determined by QPCR, normalized to cyclophilin B (PPIB)

17 Duration of MAP4K4 Silencing in vivo 2.5 Silencing of MAP4K4 persists for at least 1 week. Map4K4 Expression % P= % P= % P= Day 3 Day 5 Day 8 Day 3 Day 5 Day 8 Day 3 Day 5 Day 8 sd-rxrna injections Map4K4 sd-rxrna Nontargeting sd-rxrna PBS Day Biopsy harvest Biopsy harvest Biopsy harvest SD rats n = 2 per group with 2 injection sites/animal /treatment 6 injection sites per animal, 600 ug/dose 3 mm skin biopsies harvested on days 3, 5, and 8 RNA isolation, analysis by qpcr, normalized to β-actin P values based on comparison to the non-targeting control at the same timepoint 17

18 Therapeutic Development of sd-rxrna for Skin Indications Results to date: Efficient uptake into dermal cells following intradermal injection Significant reduction of targeted gene Duration of at least 1 week Next steps: Dose response and extended duration studies in vivo Testing of sd-rxrnas targeting therapeutically relevant genes mrna reduction Phenotypic endpoint 18

19 Phases of Repair in Acute Wound Healing Source: S Enoch and P Price, Cellular, molecular and biochemical differences in the pathophysiology of healing between acute wounds, chronic wounds and wounds in the aged, World Wide Wounds, Aug 2004, pg 3. 19