ALG-020572 and ALG-020576: Best-in-Class Potential Add your logos here Background and Aims Methods Wing Modification with Luxna 3 rd Gen BNA Improved Potency and Reduced Liver Toxicity References Financial Disclosures Reducing hepatitis B virus (HBV) S antigen (HBsAg) is key to achieving functional cure in chronic hepatitis B (CHB) patients. Antisense oligonucleotides (ASOs) are effective in reducing HBsAg in animal models and CHB patients. However, hepatotoxicity is a major side effect of ASO administration that can be exacerbated with higher affinity Locked-Nucleic Acid (LNA) modified ASOs. Next generation Bridged Nucleic Acid (BNA) and nucleobase modified monomers can reduce hepatotoxicity while maintaining efficacy. We have therefore applied these chemistries in our LNA-containing HBV targeting ASOs. ASOs with LNA and BNA chemistries were synthesized on ABI 394 and Expedite 8909 synthesizers using standard phosphoramidite chemistry. In vitro screening of LNA ASOs was carried out in HepG2.2.15 cells using a HBsAg release assay. Potent LNA-containing ASOs were chosen for conjugation with our proprietary N-Acetylgalactosamine-4 (GalNac4). BNA wing and nucleobase modifications were applied using an in-house algorithm and we compared these constructs to the all-LNA ASO parents in the adeno- associated virus (AAV)-HBV mouse model. 1) Setoguchi K. et. al. Molecular Therapy: Nucleic Acids Vol. 9 December 2017 2) Kobori T. et. al. Poster 066 OTS 2019 All authors are Aligos Therapeutics employees Jin Hong, Rajendra Pandey, Vivek K. Rajwanshi, Dinah Misner, Hua Tan, John Cortez, Yuchun Nie, Suping Ren, Dana Cho, Laxman Eltepu, Tilani De Costa, Priya Mishra, Hyunsoon Kang, Aneerban Bhattacharya, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt and Leonid N. Beigelman Aligos Therapeutics, Inc., South San Francisco, CA, USA Best in class hepatitis B virus anti-sense oligonucleotides: Next generation bridged nucleic acid chemistries significantly improve the therapeutic index by reducing hepatotoxicity and increasing in vivo efficacy in a mouse model Abstract SAT434 Conclusions A) Applying Luxna chemistries, including 3 rd generation BNA wing and nucleobase gap modifications can increase therapeutic index as demonstrated by in vivo studies in the AAV-HBV model. B) ALG-020572 and ALG-020576 are HBV ASO development candidates incorporating Luxna chemistries that target the HBV S and X regions respectively. Both compounds demonstrated enhanced in vivo potency when compared to the Ph2 clinical compound, GSK836. C) Combination of ALG-020572 (S) and ALG-020576 (X) may have multiple advantages. Results from in vitro and in vivo combination studies are pending. Figure 5. Dose-Response of ALG-020572, ALG-020576 and GSK836 in the AAV-HBV Mouse Model. All 3 oligonucleotides were dosed subcutaneously at two dose levels: 6x10mg/kg and 6x3mg/kg. All mice received 3 loading doses on Days 0, 3 and 7, then 3 maintenance doses on Days 14, 21 and 35. Overall ALG-020572 targeting the S region and ALG-020576 targeting the X region have similar in vivo potency. Both oligonucleotides demonstrate enhanced potency compared to GSK836. A) B) Aligos ASO Platform Technology GalNac4 Figure 1. ASO Platform. ASOs contain LNA as well as Luxna 3 rd generation BNA AmNA 1 and Scp BNA 2 in the wings. The gap region contains Luxna nucleobases 8-Amino A, 8-Amino G, 5-OH C and 2-Thio-T. A novel GalNac4 is attached to the ASO through a unique linker structure (5m)ScpC GalNac4 ALG-020089, 3x10 mg/kg ALG-020279, 3x10 mg/kg Vehicle C) ALG-020089, 3x10 mg/kg ALG-020279, 3x10 mg/kg Vehicle ALG-020572 and ALG-020576 are Development Candidates Containing Luxna Chemistries Figure 3. A single (2s)T substitution in the DNA Gap of ASO eliminated liver tox while maintaining potency. ALG-020090 is all LNA HBV ASO with all DNA Gap. A) ALG- 020239 is identical to ALG-020090 except that the 2 nd position of the gap contains (2s)T instead of DNA T. B) In AAV-HBV mouse model, both oligos were dosed subcutaneously 3x10mg/kg, weekly. Serial blood collections were made every 5 days and HBsAg ELISA was carried out in the serum. With nucleobase modification, in vivo potency was maintained. C) When ALT assay was conducted in the same serum, the nucleobase gap modification eliminated liver tox with maximum ALT reduced 20 times. A) B) C) (2s)T GalNac4 ALG-020090, 3x10 mg/kg ALG-020239, 3x10 mg/kg Vehicle ALG-020090, 3x10 mg/kg ALG-020239, 3x10 mg/kg Vehicle Gap Modification with Luxna Nucleobase Reduced Liver Toxicity While Maintaining Potency ALG-020572 ALG-020576 Figure 4. Aligos HBV ASO Development Candidates with Potential for Combination. ALG-020572 and ALG- 020576 target the S and X regions as shown. These can be developed individually or in combination. The combination demonstrates broader genotypic coverage, a higher resistance barrier and is less likely to lose the cleavage site in integrated HBV genomes. Genotype A B C D E F G H I J # of Clinical Isolates 1260 2306 3202 1367 376 318 134 45 103 30 ALG-020572 (S) 98% 100% 99% 100% 100% 100% 100% 100% 100% 100% ALG-020576 (X) 99% 100% 99% 100% 96% 100% 99% 100% 100% 97% ALG-020572 (S) + ALG-020576 (X) 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% Table 1. Aligos HBV ASO Development Candidates % Homology across HBV Genotypes Individually or in Combination. ALG-020572 (S) and ALG-020576 (X) show a high degree of homology with >7,774 HBV clinical isolates across Genotypes A to J as individual triggers. When combined, the % homology is 100% across all clinical isolates of all genotypes. Homology is defined as perfect match or with 1 mismatch. Loading Doses Days 0, 3 and 7 followed by 3 Weekly Maintenance Doses 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Serum HBsAg Mean ± SEM Day of study HBsAg (IU/mL) 0 5 10 15 G 01: Vehicle, SC, 6 doses G 03: ALG-020576, 6x10 mg/kg, SC G 04: ALG-020576, 6x3 mg/kg, SC G 05: ALG-020572, 6x10 mg/kg, SC G 06: ALG-020572, 6X3 mg/kg, SC G 15: GSK836, 6x10 mg/kg, SC G 16: GSK836, 6x3 mg/kg, SC 20 25 30 35 ALT levels were normal for all dose groups Figure 2. A single Scp BNA substitution in the wing of all LNA ASOs increased in vivo potency and reduced liver toxicity. ALG- 020089 is an all LNA HBV ASO. A) ALG- 020279 is identical to ALG-020089 except that the 2 nd position in the 3’ wing contains (5m)Scp C instead of LNA C. B) In the AAV- HBV mouse model, both oligonucleotides were dosed subcutaneously 3x10mg/kg, weekly. Serial blood collections were taken every 5 days and HBsAg ELISA was used to measure serum levels. With 3 rd Gen BNA chemistry, the HBsAg nadir was improved 0.5 log 10 (IU/ml). C) ALT measurement in the same samples indicated that maximum ALT was reduced 3-fold with 3 rd Gen BNA.