Aposense Molecular NanoMotors (MNMs) The first platform, energized by the membrane dipole potential, for systemic delivery of Nucleic Acids September 2017 Aposense Ltd. 5-7 Odem St., P.O.Box 7119, Petach Tikva 49170, Israel Tel: 972-73-2397-600 Fax: 972-3-9215714 www.aposense.com
CAUTIONARY NOTE REGARDING FORWARD-LOOKING STATEMENTS The following is a presentation of our Company’s business and operations. This presentation does not constitute an offer to s ell or the solicitation of an offer to subscribe for or buy or sell any of our securities. The information contained in this presentation does not constitute a basis for making an investment decision and does not constitute a recommendation or opinion, nor is it a substitute for the discretion of a potential investor. Readers are urged to carefully review and consider the various disclosures made throughout our immediate and interim reports as filed with the distribution systems of the Israeli Securities Authority (https://www.magna.isa.gov.il) and with the Tel Aviv Stock Exchange Ltd. (http://maya.tase.co.il/reports/company), which are designed to advise interested parties of the risks and factors that may affect our business, financial condition, results of operations and prospects. Certain information included or incorporated by reference in the following presentation may be deemed to be “forward - looking statements” within the meaning of the Israeli Securities Law, 5728-1968. Forward-looking statements are often characterized by the use of forward- looking terminology such as “may,” “will,” “expect,” “anticipate,” “estimate,” “continue,” “believe,” “should,” “intend,” “project” or other similar words, but are not the only way these statements are identified. Th e following presentation may contain forward-looking statements that include, but are not limited to, projections about our business and operations and/or our future revenues, expenses and profitability. These statements are only current predictions and are subject to known and unknown risks, uncertainties, and other factors that may cause our or our industry’s actual results, events, levels of activity, circumstances, performance and/or achievements to be materially different from those anticipated expressed or implied by such the forward-looking statements, due to factors that include, but are not limited to: (-) local and areal political and economic conditions; (-) changes in global and local economy and capital markets; (-) our ability to develop and bring to market new products; (-) our ability to successfully complete any necessary or required pre-clinical and/or clinical studies with our pre-clinically and/or clinically developed products; (-) our ability to receive regulatory clearance or approval to market our products and/or changes in regulatory, legislative and/or international standards environment; (-) our success in implementing our business- development, sales, marketing and/or manufacturing plans; (-) the level of adoption of our future products by medical practitioners; (-) the emergence or existence of other products that may make our products obsolete; (-) protection and validity of our patents and other intellectual property rights; (-) the effect of competition by other companies and technologies; (-) the ability to obtain reimbursement for our products from government and commercial payers; and (-) our ability to raise sufficient funds for completion of products research and development and/or our ability to partner with strategic partners and/or investors. You should not rely upon forward-looking statements as predictions of future events. We cannot guarantee future results, levels of activity, performance, or achievements reflected in the forward-looking statements. You should not put undue reliance on any forward- looking statements. Any forward-looking statements in this presentation are made as of the date hereof, and we undertake no obligation to report events or to report the occurrence of unanticipated events that may lead to the actual events, results, performance, circumstances or achievements concerning our business and operations being different than as envisaged by such forward looking statements and/or to publicly update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except as required by law. Date of presentation: September 2017. 2
Milestones in the Evolution of siRNA Therapeutics: From Discovery to the Systemic Delivery Barrier 1998 Discovery of gene silencing through injection of dsRNA to worms 2000 Discovery of the RNA Induced Silencing Complex (RISC) 2001 Discovery of the Dicer endonuclease 2001-2006 The Power of siRNA is demonstrated in numerous applications across numerous fields of medicine; Huge investments in the field by Pharma Companies 2006 Nobel Prize to Andrew Z. Fire and Craig C. Mello; "for their discovery of RNA interference - gene silencing by double- stranded RNA” The Systemic Delivery Barrier: Large withdrawal from the field: “ siRNA is 2006 - 2014 probably non-druggable ” 2014 First demonstration of clinical benefit of siRNA targeted to the liver (ATTR amyloidosis) 2014 - Renewed interest in siRNA; Strategic alliances Only for liver-related indications; or for topical administration • Recognition that siRNA may hold a great therapeutic promise 2014 - • Pending on solving The Systemic Delivery Barrier Lack of efficacious and Universal Tools for Systemic Delivery of siRNA; Widely-Recognized as The Major Factor that Holds-Back Clinical Implementation of siRNA
siRNA therapeutics; The promise and the unmet need “ The siRNA Technology is quite amazing, to be sure. These small pieces of nucleic acid are capable of knocking down genes with extremely high selectivity” “The most intractable problem with RNAi therapeutics has to do with systemic delivery . Nucleic acids such as RNAi are relatively large and carry a negative charge, preventing absorption through the cell membrane …. Considerable efforts have been expended breaking down the delivery barrier. Yet even the combined resources of both Big Pharma and small biotechs have not been enough to solve the problem, at least not yet”. Pfizer 4
siRNA: The delivery challenge Classical drug Genetic drug Aspirin: Molecular weight 180 Daltons siRNA: Molecular weight 14,000 Daltons 42 negative charges 5
Delivery of siRNA across cell membranes into cells requires a very large activation energy 6 4
Apo-Si Molecular Nanomotor Platform A disruptive technology Innovative A novel internal Molecular powerhouse NanoMotors Breaking-down the membrane delivery barrier 7
The membrane dipole potential • Electric voltage measured between: Membrane / water interface (-) membrane center (+) • Generated by the ordered carbonyl groups of the phospholipids A novel internal • Environment: very hydrophobic powerhouse • Dielectric constant: 2-4 • Due to hydrophobic environment, results in an enormously strong intra-membrane electric field of 10 8 -10 9 V/m !!! ΔΨ = the trans-membrane potential Ψ s = the surface potential Ψ d = the dipole potential ε = the dielectric constant 8 Wang L. Annual Review of Biochemistry 81: 615-635, 2012 6
The Apo-Si Molecular NanoMotor (MNM): Optimized molecular structure for utilizing the membrane dipole potential for drug delivery Point for “click attachment” to any Oligonucleotide sequence (DNA or RNA) Innovative Molecular NanoMotors Detachment Transmission Electrical Unit Unit Unit • Small-chemical Entity • Proprietary; All in-house IP • Non Toxic • Modular 9
The MNM attachment module • Standard building block of any oligonucleotide synthesis • The locations and number of moieties can be tailored per the specific application Detachment Module 10
The dsiRNA-MNM construct Breaking-down the membrane delivery barrier Prolonged residence in body fluids Membrane surface Trans-membrane delivery to reach the cytoplasm MNM; MW ≈ 700 Daltons dsiRNA ; MW ≈ 14,000 Daltons 11
Endocytosis; Flip-flop; RNA detachment Endosome-mediated Direct entry entry Flip-flop Cytoplasm MNM: • Detachment from RNA • Excretion: Kidneys / Liver RNA: Interaction with Dicer & RISC Gene Silencing 12
Robust trans-membrane delivery of Apo-Si-MNM Conjugates; B16 melanoma cells Control MNM-dsiRNA Conjugate Control: fluorescently-labeled Fluorescently-labeled dsiRNA without MNMs dsiRNA, conjugated to MNMs Cy3= Red fluorophore X 20 11
Delivery of dsDNA, Attached to Two Apo-Si-MNMs Uptake of Apo-Si-MNM Conjugate is rapid and efficient; 3T3 cells; Time course; 40nM; FACS 3T3 Cells 97 100 90 90 78 80 % Positive Events, Cy3 64 70 60 50 40 30 20 10 1 0 15 30 1 2 control min min hour hours N=3; n=6 12 Mean ± SE
Shutting-down the internal membrane electrical field inhibits delivery of Apo-Si-MNMs Conjugate Complete inhibition by Phloretin FACS analysis; Phloretin 750µM 3hr, Apo-Si-MNMs Conjugate 40nM, 2hr Field OFF Field ON Field ON + MNMs No MNMs + MNMs Control Apo-Si-MNMs Apo-Si-MNMs Conjugate + Conjugate Phloretin 0% 1% 95% Apo-Si Delivery; (Cy3) Apo-Si Delivery; (Cy3) Apo-Si Delivery; (Cy3) EGFP fluorescence EGFP fluorescence EGFP fluorescence 15
Shutting-down the membrane dipole potential inhibits delivery of Apo-Si-MNMs Conjugate; dose-related FACS analysis; 3T3 cells; Phloretin 3hr, Apo-Si-MNMs Conjugate 40nM 2hr N=3; n=6 16 Mean ± SE
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