Editorial: In vitro mechanistic evaluation of nucleic acid polymers: A cautionary tale

Abstract

Nucleic acid polymers (NAPs) are broad-spectrum antiviral compounds active against diverse enveloped viruses and other infectious agents.1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar The activity of all NAPs is sequence independent and driven by a length-dependent (optimal with 40-mers) and phosphorothioation (hydrophobic)-dependent interaction with the exposed hydrophobic surfaces of amphipathic alpha helices.1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar,2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar Recent focus on NAPs for the treatment of chronic hepatitis B and hepatitis D infection has been driven by several phase II trials demonstrating the ability of several different clinical NAP compounds (REP 2055, REP 2139, and REP 2165) to achieve rapid hepatitis B surface antigen (HBsAg) loss and seroconversion and high rates of therapeutic transaminase flares, leading to high rates of functional cure of hepatitis B virus (HBV) and hepatitis D virus (HDV).2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar, 3Bazinet M. Pantea V. Placinta G. Moscalu I. Cebotarescu V. Cojuhari L. Jimbei P. Iarovoi L. Smesnoi V. Musteata T. et al.Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naive to nucleos(t)ide therapy.Gastroenterology. 2020; 158: 2180-2194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 4Bazinet M. Pantea V. Cebotarescu V. Cojuhari L. Jimbei P. Anderson M. Gersch J. Holzmayer V. Elsner C. Krawczyk A. et al.Persistent control of HBV and HDV infection following REP 2139-Ca and pegIFN therapy in chronic HBV/HDV co-infection.Hepatol. Commun. 2020; 5: 189-202Crossref PubMed Scopus (20) Google Scholar Recently published in vitro data5Kao C.C. Nie Y. Ren S. Tilani N. De Costa T.S. Pamdey R.K. Hong J. Smith D.B. Symons J.,A. Beigelman L. et al.Mechanism of action of hepatitis B virus S antigen transport-inhibiting oligonucleotide polymer, STOPS, molecules.Mol. Ther. Nucleic Acids. 2021; 27: 335-348Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar suggest that NAPs modified with locked nucleic acids (LNAs) have substantially improved potency compared with previously published NAPs and suggested mechanisms of action and potential host targets very different from those previously published for NAPs. The even more recent announcement of the abandonment of the LNA-modified NAP ALG-10133 and the entire LNA-modified NAP platform because of a lack of antiviral activity in humans6AligosAligos Halting Further Development of STOPS™ Drug Candidate, ALG-010133.2022Google Scholar raises serious questions regarding these recently published in vitro data. The disconnect between the efficacy of LNA-modified NAPs reported in vitro and in the clinic are the result of several limitations/artifacts in the experimental approaches used that are important to highlight. REP 2055 is a 40-mer phosphorothioate oligodeoxyribonucleotide with the sequence (AC)20. REP 2139 is an RNA derivative of REP 2055 in which each ribose is 2′O methylated and each cytosine base is 5-methylated (Figure 1). The poly AC sequence does affect the antiviral activity of NAPs but is designed to eliminate intra-/intermolecular interactions, to minimize secondary structure and off-target interactions, and to minimize recognition by pattern recognition receptors to render NAPs immunologically inert while maintaining the flexible B-form DNA structure required for optimal NAP activity.1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar Both REP 2055 and REP 2139 have been extensively evaluated against HBV infection in vitro, in vivo, and in human studies, in which they have been shown to have comparable activity2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar demonstrating that 2′ribose modification in the NAP polymer has no impact on the antiviral activity of NAPs. ALG-10000 is a previously described,8Vaillant, A, and Juteau, JM. Antiviral Oligonucleotides. US Patent: 7,358,068.Google Scholar LNA-modified derivative of REP 2055 and REP 2139 (Figure 1). The implications of LNA modification on NAP activity are discussed below. REP 2031 is a 40-mer poly C NAP (Figure 1) that has broad-spectrum antiviral activity in vitro and in vivo against all enveloped viruses and other infectious agents except HBV.2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar Like all polypyridines, REP 2031 forms intermolecular tetramers at pH < 6.8 (Figure 2),9Kanehara H. Mizuguchi M. Tajima K. Kanaori K. Makino K. Spectroscopic evidence for the formation of four-stranded solution structure of oligodeoxycytidine phosphorothioate.Biochemistry. 1997; 36: 1790-1797Crossref PubMed Scopus (47) Google Scholar a transformation that does not occur with purine/pyrimidine altimers such as REP 2055, REP 2139, and REP 216510Geinguenaud F. Liquier J. Brevnov M.G. Petrauskene O.V. Alexeev Y.I. Gromova E.S. Taillandier E. Parallel self-associated structures formed by T,C-rich sequences at acidic pH.Biochemistry. 2000; 39: 12650-12658Crossref PubMed Scopus (18) Google Scholar or degenerate NAPs such as REP 2006 and REP 2107 (Figures 1 and 2). The tetramerization of REP 2031 prevents target engagement at acidic pH and leads to the inactivity of REP 2031 against HBV infection in vitro7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,11Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.Antimicrob. Agents Chemother. 2013; 57: 5291-5298Crossref PubMed Scopus (42) Google Scholar and in vivo.12Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo.Antimicrob. Agents Chemother. 2013; 57: 5299-5306Crossref PubMed Scopus (46) Google Scholar These observations demonstrate that NAPs that are active against HBV infection act inside the acidified lumen of secretory vesicles. Therefore, host targets for NAPs need to satisfy two criteria: (1) be present and active inside secretory vesicles and (2) have binding interactions that are consistent with length and phosphorothioate-dependent antiviral activity conserved in all NAPs. Although the ligand-mediated approach used for identifying NAP targets described by Kao et al. is in principle correct, the methodology they used suffers from several critical flaws. The first is the use of ALG-10000 as the single and only NAP bait in the ligand assay. Several kinds of off-target protein interactions can occur with NAPs: off-target hydrophilic versus antiviral hydrophobic and small-target versus large-target (antiviral) interface-driven interactions.2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar These off-target interactions will be stronger with ALG-10000 because of the absence of 2′O methylation, a modification that increases the hydration of oligonucleotides and inhibits off-target interactions13Yoo B.H. Bochkareva E. Bochkarev A. Mou T.C. Gray D.M. 2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro.Nucleic Acids Res. 2004; 32: 2008-2016Crossref PubMed Scopus (68) Google Scholar,14Egli M. Pallan P.S. Insights from crystallographic studies into the structural and pairing properties of nucleic acid analogs and chemically modified DNA and RNA oligonucleotides.Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 281-305Crossref PubMed Scopus (52) Google Scholar and does not affect the antiviral activity of NAPs.1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar,2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar Additionally, LNA-modified oligonucleotides adopt a rigid A-form DNA structure,15Dash C. Marino J.P. Le Grice S.F. Examining Ty3 polypurine tract structure and function by nucleoside analog interference.J. Biol. Chem. 2006; 281: 2773-2783Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 16Kierzek E. Pasternak A. Pasternak K. Gdaniec Z. Yildirim I. Turner D.H. Kierzek R. Contributions of stacking, preorganization, and hydrogen bonding to the thermodynamic stability of duplexes between RNA and 2'-O-methyl RNA with locked nucleic acids.Biochemistry. 2009; 48: 4377-4387Crossref PubMed Scopus (41) Google Scholar, 17Pabon-Martinez Y.V. Xu Y. Villa A. Lundin K.E. Geny S. Nguyen C.H. Pedersen E.B. Jorgensen P.T. Wengel J. Nilsson L. et al.LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures.Sci. Rep. 2017; 7: 11043Crossref PubMed Scopus (22) Google Scholar compared with the flexible B-form DNA structure of non-LNA-modified NAPs, which will also affect binding affinity and selectivity. Although the protein targets identified by Kao et al. bind to ALG-10000 ex vitro, and RNAi-mediated reduction of these targets results in inhibition of HBsAg secretion, these cannot be identified as physiological NAP targets involved in HBV activity without controlling for binding driven by off-target hydrophilic interactions (i.e., by using an ALG-10000 analog without the phosphorothioate modification) or for off-target smaller interface interactions (by using an ALG-10000 analog with suboptimal size, such as a 20-mer) or for pH-dependent interactions by performing the binding at neutral and acidic pH or for the effect of the LNA structural alterations by using REP 2055. Importantly, none of the identified targets reported by Kao et al. exhibits the hallmark binding site for NAPs,1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar,2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar and with the exception of GRP78, none is found within the lumen of acidified vesicles. Moreover, if RPLP1 and RPLP2 (60S ribosomal subunits), SRSF1 (pre-mRNA splicing factor), and HNRNPA2B1 (nuclear ribonucleoprotein) as identified by Kao et al. were indeed physiological targets for NAPs, the well-conserved nature of these housekeeping genes in all mammals would result in the antiviral activity of NAPs in rodent models of HBV infection being easily observed, which is not the case, even with prolonged NAP therapy with REP 2055 or REP 2139.18Schoneweis K. Motter N. Roppert P.L. Lu M. Wang B. Roehl I. Glebe D. Yang D. Morrey J.D. Roggendorf M. et al.Activity of nucleic acid polymers in rodent models of HBV infection.Antivir. Res. 2018; 149: 26-33Crossref PubMed Scopus (11) Google Scholar Additionally, prolonged interference with the housekeeping functions of these proteins with NAP therapy would be expected to result in significant toxicity in animals and humans, which has never been observed with any clinically evaluated NAP.3Bazinet M. Pantea V. Placinta G. Moscalu I. Cebotarescu V. Cojuhari L. Jimbei P. Iarovoi L. Smesnoi V. Musteata T. et al.Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naive to nucleos(t)ide therapy.Gastroenterology. 2020; 158: 2180-2194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20Al-Mahtab M. Bazinet M. Vaillant A. Safety and efficacy of nucleic acid polymers in monotherapy and combined with immunotherapy in treatment-naive Bangladeshi patients with HBeAg+ chronic hepatitis B infection.PLoS One. 2016; 11: e0156667Crossref PubMed Scopus (127) Google Scholar, 21Bazinet M. Pantea V. Cebotarescu V. Cojuhari L. Jimbei P. Albrecht J. Schmid P. Le Gal F. Gordien E. Krawczyk A. et al.Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial.Lancet Gastroenterol. Hepatol. 2017; 2: 877-889Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar An ongoing study to identify the physiological host target(s) of NAP interaction involved in the antiviral activity against HBV is nearing completion with REP 2139,22Boulon R. Angelo L. Blanchet M. Vaillant A. Labonte P. PH-dependent interaction of NAPs with the HSP40 chaperone DnaJB12.Hepatology. 2021; 74: 512AGoogle Scholar which includes controlled ligand binding done at neutral and acidic pH and comparing binding interactions observed with REP 2139 with those observed with no-PS (REP 2147) and 20-mer analogs (REP 2179) of REP 2139 and also the acid-inactivated REP 2031. Preliminary results have clearly excluded all of the targets identified by Kao et al. and identified the HSP40 chaperone DnaJB12 as the physiological target for all NAPs acting in HBV infection. Pharmacological effects of phosphorothioate oligonucleotides such as antisense compounds are easily achieved in the liver in vivo and in humans because of the natural uptake and cytoplasmic transit of these compounds in hepatocytes (Figure 3A ). However, standard treatment of hepatocyte-derived cell lines with antisense compounds results in endosomal entrapment of oligonucleotides (Figure 3A), preventing cytoplasmic trafficking and pharmacological effects of this chemical class of compounds.7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,23Yang B. Ming X. Cao C. Laing B. Yuan A. Porter M.A. Hull-Ryde E.A. Maddry J. Suto M. Janzen W.P. et al.High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.Nucleic Acids Res. 2015; 43: 1987-1996Crossref PubMed Scopus (57) Google Scholar,24Koller E. Vincent T.M. Chappell A. De S. Manoharan M. Bennett C.F. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.Nucleic Acids Res. 2011; 39: 4795-4807Crossref PubMed Scopus (209) Google Scholar With the exception of primary co-cultures of duck liver parenchymal and non-parenchymal cells,11Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.Antimicrob. Agents Chemother. 2013; 57: 5291-5298Crossref PubMed Scopus (42) Google Scholar NAPs also obey this limitation, with post-entry pharmacological effects against HBV infection easily obtained in vivo and in humans2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar but not observed in HepG2.2.15, HepaRG, Huh-106, and primary human hepatocytes with standard treatment.25Guillot C. Martel N. Berby F. Bordes I. Hantz O. Blanchet M. Sureau C. Vaillant A. Chemin I. Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes.PLoS One. 2017; 12: e0179697Crossref PubMed Scopus (20) Google Scholar,26Beilstein F. Blanchet M. Vaillant A. Sureau C. Nucleic acid polymers are active against hepatitis delta virus infection in vitro.J. Virol. 2018; 92 (e01416-17)Crossref PubMed Scopus (29) Google Scholar In order to overcome this limitation with oligonucleotide treatment in vitro, it is tempting to use transfection. As described by Kao et al., transfection leads to the enhanced delivery of oligonucleotides to the cytoplasm; however, although this effect is useful for studying the activity of oligonucleotides that act in the cytoplasm (i.e., antisense or RNAi), it is not appropriate for the evaluation of NAPs, as the action for these compounds occurs in the secretory pathway (Figures 3A and 3B). Additionally and equally important is the unsuitability of transfection to evaluate the relative in vitro activity of NAPs with significant differences in hydration or secondary structure. The disconnect between the in vitro activity of a variety of NAPs with activity validated in vivo and in humans observed with transfection and in the absence of transfection/in vivo has been previously observed in several independent laboratories in collaborations that began in 2007 (A.Vaillant, unpublished data) and are summarized in Figure 2. Liposome formation is critical to ensure efficient oligonucleotide delivery with lipid-based transfection and is inhibited by oligonucleotide hydration27Akhtar S. Basu S. Wickstrom E. Juliano R.L. Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).Nucleic Acids Res. 1991; 19: 5551-5559Crossref PubMed Google Scholar,28Jaaskelainen I. Monkkonen J. Urtti A. Oligonucleotide-cationic liposome interactions. A physicochemical study.Biochim. Biophys. Acta. 1994; 1195: 115-123Crossref PubMed Scopus (101) Google Scholar (Figure 3B). Thus, poorly hydrated NAPs such as REP 2055 and ALG-10000 perform well via transfection, whereas NAPs well hydrated with complete 2′O methylation such as REP 2139 and REP 2107 perform poorly (see Figure 2), even though they have comparable antiviral activity to REP 2055 when evaluated in vitro with non-transfection-dependent systems, in vivo, and in humans.7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,29Noordeen F. Scougall C.A. Grosse A. Qiao Q. Ajilian B.B. Reaiche-Miller G. Finnie J. Werner M. Broering R. Schlaak J.F. et al.Therapeutic antiviral effect of the nucleic acid polymer REP 2055 against persistent duck hepatitis B virus infection.PLoS One. 2015; 10: e0140909Crossref PubMed Scopus (56) Google Scholar,30Quinet J. Jamard C. Burtin M. Lemasson M. Guerret S. Sureau C. Vaillant A. Cova L. Nucleic acid polymer REP 2139 and nucleos(T)ide analogues act synergistically against chronic hepadnaviral infection in vivo in Pekin ducks.Hepatology. 2018; 67: 2127-2140Crossref PubMed Scopus (18) Google Scholar Thus, the differences in apparent in vitro activity against HBV between LNA-modified NAPs (e.g., ALG-10000) and non-LNA-modified NAPs (i.e., REP 2139) are a function of reduced transfection efficiency with REP 2139, not a lower specific antiviral activity. Additionally, degenerate NAPs such as REP 2006 and REP 2107 adopt secondary structure at neutral pH (Figure 4A ), but protonation of these NAPs at acidic pH abolishes this secondary structure (Figures 2 and 4B), which renders these NAPs active in vitro and in vivo. Although these NAPs have similar antiviral activity in vitro in the absence of transfection and in vivo compared with REP 2055 and REP 2139,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,11Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.Antimicrob. Agents Chemother. 2013; 57: 5291-5298Crossref PubMed Scopus (42) Google Scholar,12Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo.Antimicrob. Agents Chemother. 2013; 57: 5299-5306Crossref PubMed Scopus (46) Google Scholar they have no antiviral effect in vitro with lipid-based transfection (Figure 2). This also demonstrates that oligonucleotides with significant secondary structure are also poorly transfected (Figure 3B). The use of the in vivo-validated positive NAP controls REP 2006, REP 2107, REP 2055, and REP 2139 and the validated negative controls REP 2031 and phosphodiester versions of REP 2107 (REP 2086) and REP 2139 (REP 2147) are critical to ensure that any in vitro system is appropriate for evaluating NAP activity.7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar These important controls are missing from transfection studies described in Kao et al. and would have revealed these defects in transfection-based evaluation of NAP activity. A key observation illustrating altered NAP trafficking with transfection is the observation of apparent antiviral activity of REP 2031 against HBV infection when transfected in vitro (Figure 2). This observation indicates non-physiological cytoplasmic target interactions occurring under these experimental conditions. When used in in vitro models in which normal oligonucleotide trafficking occurs in the absence of transfection11Noordeen F. Vaillant A. Jilbert A.R. Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.Antimicrob. Agents Chemother. 2013; 57: 5291-5298Crossref PubMed Scopus (42) Google Scholar or when endosomal entrapment is removed,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar REP 2031 activity is absent or significantly reduced compared with REP 2006, REP 2055, or REP 2139. In our ongoing studies,22Boulon R. Angelo L. Blanchet M. Vaillant A. Labonte P. PH-dependent interaction of NAPs with the HSP40 chaperone DnaJB12.Hepatology. 2021; 74: 512AGoogle Scholar we have identified casein kinase 1 delta as a non-physiological target for NAPs in the cytoplasm.22Boulon R. Angelo L. Blanchet M. Vaillant A. Labonte P. PH-dependent interaction of NAPs with the HSP40 chaperone DnaJB12.Hepatology. 2021; 74: 512AGoogle Scholar Engagement of this target appears to universally inhibit retrograde vesical transport, yielding inhibition of both HBsAg and hepatitis B e-antigen (HBeAg) secretion.22Boulon R. Angelo L. Blanchet M. Vaillant A. Labonte P. PH-dependent interaction of NAPs with the HSP40 chaperone DnaJB12.Hepatology. 2021; 74: 512AGoogle Scholar It is important to note that NAPs do not affect HBeAg secretion in vitro7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar or in humans (A. Vaillant, personal communication). Although not reported by Kao et al. in their publication, the authors also performed transfection experiments with an LNA-modified version of REP 2031 (ALG-10202; Figure 1),31Fitzgerald M. Hong J. Pandey R. Rajwanshi V. Tan H. Nie Y. Kao C.C. Ren S. Cortez J. Smith D.B. et al.Structural requirements for S-antigen transport-inhibiting oligonucleotide polymer inhibition of hepatitis B surface antigen secretion.J. Hepatol. 2020; 73: S876-S877Abstract Full Text PDF Google Scholar in which they observed no activity against HBV in vitro. This is easily explained by the spontaneous formation of highly stable alpha-helical structures of LNA-modified polycytidine oligonucleotides at neutral pH17Pabon-Martinez Y.V. Xu Y. Villa A. Lundin K.E. Geny S. Nguyen C.H. Pedersen E.B. Jorgensen P.T. Wengel J. Nilsson L. et al.LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures.Sci. Rep. 2017; 7: 11043Crossref PubMed Scopus (22) Google Scholar,32Xu Y. Gissberg O. Pabon-Martinez Y.V. Wengel J. Lundin K.E. Smith C.I.E. Zain R. Nilsson L. Villa A. The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: a molecular simulation and binding study.PLoS One. 2019; 14: e0211651PubMed Google Scholar (Figure 2), which most likely block liposome formation. UNC 7938 is a small molecule that restores endosomal release of oligonucleotides in vitro.23Yang B. Ming X. Cao C. Laing B. Yuan A. Porter M.A. Hull-Ryde E.A. Maddry J. Suto M. Janzen W.P. et al.High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.Nucleic Acids Res. 2015; 43: 1987-1996Crossref PubMed Scopus (57) Google Scholar Brief exposure of HepG2.2.15 cells to UNC 7938 following standard NAP treatment led to evacuation of NAPs from endosomes, with subsequent cytoplasmic trafficking.7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar Using this approach, post-entry activity of NAPs could be observed that recapitulated the antiviral effects of NAPs previously observed in vivo and in humans.7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,33Boulon R. Blanchet M. Lemasson M. Vaillant A. Labonte P. Characterization of the antiviral effects of REP 2139 on the HBV lifecycle in vitro.Antivir. Res. 2020; 5: 189-202Google Scholar These effects included (1) size and phosphorothioate-dependent antiviral activity; (2) comparable antiviral activity of non-hydrated (REP 2055) and hydrated (REP 2139) NAPs, recapitulating the sugar modification-independent activity observed in vivo and in humans; and (3) comparable activity of NAPs with a degenerate sequence (REP 2107). Importantly, REP 2031 demonstrated reduced activity in this model system,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar consistent with the absence of antiviral activity of this NAP in vivo, demonstrating that trafficking of NAPs through the acidified secretory pathway was occurring. Additional validation of this system included the observation of selective effects on the assembly and secretion or subviral particles without affecting the secretion of HBeAg or virions,33Boulon R. Blanchet M. Lemasson M. Vaillant A. Labonte P. Characterization of the antiviral effects of REP 2139 on the HBV lifecycle in vitro.Antivir. Res. 2020; 5: 189-202Google Scholar consistent with the antiviral effects of NAPs observed in vivo and in humans. REP 2165 is an analog of REP 2139 that is designed to be more susceptible to endonuclease attack at three unmodified riboadenosines19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar (Figure 1). ALG-10133 is an LNA-modified analog of REP 2165 in which every other 2′O methyl ribose 5-methylcytidine is replaced by LNA 5-methylcytidine. Additionally, ALG-10133 contains four unmodified riboadenosine breaks compared with the three present in REP 2165 (Figure 1). Although REP 2165 is comparable in activity to REP 2055 and REP 2139,1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar,3Bazinet M. Pantea V. Placinta G. Moscalu I. Cebotarescu V. Cojuhari L. Jimbei P. Iarovoi L. Smesnoi V. Musteata T. et al.Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naive to nucleos(t)ide therapy.Gastroenterology. 2020; 158: 2180-2194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar ALG-10133 had no significant effect on HBsAg reduction in human HBV infection at doses at which REP 2055 and REP 2139 yield 4–7 log reductions from baseline in HBsAg.6AligosAligos Halting Further Development of STOPS™ Drug Candidate, ALG-010133.2022Google Scholar The reasons the failure of ALG-10133 are threefold: first, the structural alterations that occur in LNA-modified oligonucleotides have the effect of reducing their hydration.34Kaur H. Arora A. Wengel J. Maiti S. Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes.Biochemistry. 2006; 45: 7347-7355Crossref PubMed Scopus (133) Google Scholar,35Pande V. Nilsson L. Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations.Nucleic Acids Res. 2008; 36: 1508-1516Crossref PubMed Scopus (51) Google Scholar As such, in ALG-10133, the doping of the uniform 2′O-methylation present in REP 2165 (Figure 1) substantially reduced the hydration of this NAP and allowed it to be efficiently transfected, yielding apparent (but artifactually) increased antiviral activity by transfection relative to REP 2139,31Fitzgerald M. Hong J. Pandey R. Rajwanshi V. Tan H. Nie Y. Kao C.C. Ren S. Cortez J. Smith D.B. et al.Structural requirements for S-antigen transport-inhibiting oligonucleotide polymer inhibition of hepatitis B surface antigen secretion.J. Hepatol. 2020; 73: S876-S877Abstract Full Text PDF Google Scholar similar to ALG-10000.5Kao C.C. Nie Y. Ren S. Tilani N. De Costa T.S. Pamdey R.K. Hong J. Smith D.B. Symons J.,A. Beigelman L. et al.Mechanism of action of hepatitis B virus S antigen transport-inhibiting oligonucleotide polymer, STOPS, molecules.Mol. Ther. Nucleic Acids. 2021; 27: 335-348Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar The second reason for the failure of ALG-10133 is the structural rigidity LNA imparts to the NAP. The unstructured and flexible nature of NAPs is essential for their optimal antiviral effect1Vaillant A. Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.Antivir. Res. 2016; 133: 32-40Crossref PubMed Scopus (89) Google Scholar,2Vaillant A. Rep 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection.ACS Infect Dis. 2019; 5: 675-687Crossref PubMed Scopus (43) Google Scholar,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar because this flexibility allows optimal interaction with uncomplexed amphipathic alpha helices, for example in the J-domain of DNAJB12 (Figure 5). As such, LNA-modified NAPs such as ALG-10000 and ALG-10133, although very structurally similar to REP 2055 and REP 2139, will have drastically reduced target engagement because of their rigidity15Dash C. Marino J.P. Le Grice S.F. Examining Ty3 polypurine tract structure and function by nucleoside analog interference.J. Biol. Chem. 2006; 281: 2773-2783Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 16Kierzek E. Pasternak A. Pasternak K. Gdaniec Z. Yildirim I. Turner D.H. Kierzek R. Contributions of stacking, preorganization, and hydrogen bonding to the thermodynamic stability of duplexes between RNA and 2'-O-methyl RNA with locked nucleic acids.Biochemistry. 2009; 48: 4377-4387Crossref PubMed Scopus (41) Google Scholar, 17Pabon-Martinez Y.V. Xu Y. Villa A. Lundin K.E. Geny S. Nguyen C.H. Pedersen E.B. Jorgensen P.T. Wengel J. Nilsson L. et al.LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures.Sci. Rep. 2017; 7: 11043Crossref PubMed Scopus (22) Google Scholar (Figure 5). Finally, early preclinical data showed that the hepatic accumulation of ALG-10133 was approximately 5-fold lower than REP 2165 at equivalent dose19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,36Hong J. Pandey R. Rajwanshi V. Tan H. Nie Y. Kao C.C. Cortez J. Misner J. Chanda S. Smith D.B. et al.S-antigen traffic-inhibiting oligonucleotide polymers (STOPS) can effectively inhibit hepatitis B surface antigen (HBsAg) secretion from hepatitis B virus (HBV) cell lines.Hepatology. 2019; 70: 430AGoogle Scholar which in the case of ALG-10133 was below the liver accumulation threshold required for antiviral activity of NAPs against HBV.19Roehl I. Seiffert S. Brikh C. Quinet J. Jamard C. Dorfler N. Lockridge J.A. Cova L. Vaillant A. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy.Mol. Ther. Nucleic Acids. 2017; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Although the reason for this suboptimal accumulation is unclear, it may be due to decreased nuclease stability or increased off-target interactions driven by the LNA modification. The correct identification of the mechanism of action of NAPs as well as their host target(s) is important given their potent antiviral effects in HBV and HDV infection in humans. However, in vitro evaluation of NAPs cannot be conducted with transfection-based approaches, because of the impact of oligonucleotide structure and hydration on liposome formation and the non-physiological accumulation of NAPs in the cytoplasm. These artifacts prevent the correct comparison of relative antiviral activity between NAPs with different modifications and result in the observation of many inhibitory effects in vitro that do not occur in vivo or in humans. The disconnect between transfection data presented with NAPs in vitro5Kao C.C. Nie Y. Ren S. Tilani N. De Costa T.S. Pamdey R.K. Hong J. Smith D.B. Symons J.,A. Beigelman L. et al.Mechanism of action of hepatitis B virus S antigen transport-inhibiting oligonucleotide polymer, STOPS, molecules.Mol. Ther. Nucleic Acids. 2021; 27: 335-348Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar,31Fitzgerald M. Hong J. Pandey R. Rajwanshi V. Tan H. Nie Y. Kao C.C. Ren S. Cortez J. Smith D.B. et al.Structural requirements for S-antigen transport-inhibiting oligonucleotide polymer inhibition of hepatitis B surface antigen secretion.J. Hepatol. 2020; 73: S876-S877Abstract Full Text PDF Google Scholar,36Hong J. Pandey R. Rajwanshi V. Tan H. Nie Y. Kao C.C. Cortez J. Misner J. Chanda S. Smith D.B. et al.S-antigen traffic-inhibiting oligonucleotide polymers (STOPS) can effectively inhibit hepatitis B surface antigen (HBsAg) secretion from hepatitis B virus (HBV) cell lines.Hepatology. 2019; 70: 430AGoogle Scholar and in humans3Bazinet M. Pantea V. Placinta G. Moscalu I. Cebotarescu V. Cojuhari L. Jimbei P. Iarovoi L. Smesnoi V. Musteata T. et al.Safety and efficacy of 48 Weeks REP 2139 or REP 2165, tenofovir disoproxil, and pegylated interferon alfa-2a in patients with chronic HBV infection naive to nucleos(t)ide therapy.Gastroenterology. 2020; 158: 2180-2194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,6AligosAligos Halting Further Development of STOPS™ Drug Candidate, ALG-010133.2022Google Scholar,20Al-Mahtab M. Bazinet M. Vaillant A. Safety and efficacy of nucleic acid polymers in monotherapy and combined with immunotherapy in treatment-naive Bangladeshi patients with HBeAg+ chronic hepatitis B infection.PLoS One. 2016; 11: e0156667Crossref PubMed Scopus (127) Google Scholar is a clear validation of these artifacts. Future evaluations of NAPs in vitro must use suitable systems as described above, which have been validated with known control NAPs,7Blanchet M. Sinnathamby V. Vaillant A. Labonte P. Inhibition of HBsAg secretion by nucleic acid polymers in HepG2.2.15cells.Antivir. Res. 2019; 164: 97-105Crossref PubMed Scopus (34) Google Scholar,33Boulon R. Blanchet M. Lemasson M. Vaillant A. Labonte P. Characterization of the antiviral effects of REP 2139 on the HBV lifecycle in vitro.Antivir. Res. 2020; 5: 189-202Google Scholar and target identification must be performed under well-controlled conditions.22Boulon R. Angelo L. Blanchet M. Vaillant A. Labonte P. PH-dependent interaction of NAPs with the HSP40 chaperone DnaJB12.Hepatology. 2021; 74: 512AGoogle Scholar The LNA modification of NAPs was first described in 20038Vaillant, A, and Juteau, JM. Antiviral Oligonucleotides. US Patent: 7,358,068.Google Scholar but discarded early in development for its negative impact on NAP flexibility and well-documented hepatotoxicity.37Swayze E.E. Siwkowski A.M. Wancewicz E.V. Migawa M.T. Wyrzykiewicz T.K. Hung G. Monia B.P. Bennett C.F. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.Nucleic Acids Res. 2007; 35: 687-700Crossref PubMed Scopus (286) Google Scholar, 38Kasuya T. Hori S. Watanabe A. Nakajima M. Gahara Y. Rokushima M. Yanagimoto T. Kugimiya A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.Sci. Rep. 2016; 6: 30377Crossref PubMed Scopus (57) Google Scholar, 39Burel S.A. Hart C.E. Cauntay P. Hsiao J. Machemer T. Katz M. Watt A. Bui H.H. Younis H. Sabripour M. et al.Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.Nucleic Acids Res. 2016; 44: 2093-2109Crossref PubMed Google Scholar To date, REP 2139 represents the fully optimized potential of this class of compounds, being highly stable, composed only of naturally occurring sugar and base modifications, and devoid of immunostimulatory properties, all while retaining its antiviral activity. I would like to acknowledge Michael Roggendorf, Camille Sureau, Lucyna Cova, and Isabelle Chemin for their collaborative efforts in the mechanistic evaluations of nucleic polymers. A.V. is an employee and shareholder of Replicor Inc.