siRNA Goes Public Again: CITY vs Alnylam and the In Vivo Models Behind the Next Wave

City Therapeutics debuted on Nasdaq on October 16, 2026, closing an upsized $230 million IPO three days later despite being just three years old, with no product revenue and a lead asset in Phase 1 ^[1]. Weeks earlier, ADARx raised approximately $446 million with its most advanced program in Phase 3 ^[2]. Together, these offerings suggest renewed investor appetite for RNAi companies across development stages.

Investor confidence, however, is not clinical validation. Next-generation RNAi therapies face two key challenges: productive delivery beyond the liver and preclinical testing against the relevant human target sequence. Alnylam and City emphasize different approaches to RNAi trigger engineering and tissue-directed delivery. Comparing their strategies highlights a shared need for models that distinguish delivery limitations from insufficient activity against the human target.

Alnylam's siRNA Platform: GalNAc Delivery and Extrahepatic Expansion

Alnylam helped establish the modern GalNAc-siRNA platform, combining extensive 2′ chemical modifications and selected phosphorothioate linkages with a trivalent GalNAc ligand that enables ASGPR-mediated uptake into hepatocytes ^[3]. Several GalNAc-siRNA medicines support dosing intervals measured in months, although schedules vary by product. Alnylam reported global net product revenues of $1.172 billion in the second quarter of 2026 across its commercial portfolio ^[4]. Alongside that commercial scale, the company continues to refine siRNA design and chemistry through its platforms.

Figure 1. RNAi therapeutics utilizing GalNAc conjugate technology ^[6].

For extrahepatic applications, Alnylam combines siRNA chemistry optimization with delivery approaches tailored to the target tissue. A prominent example is the lipophilic C16 (2′-O-hexadecyl) conjugate reported in Nature Biotechnology in 2022 ^[5]. In preclinical studies, C16-conjugated siRNAs produced potent, durable silencing in the CNS, eye and lung following local administration ^[5].

Figure 2. RNAi therapeutics utilizing C16 conjugates technology ^[6].

Mivelsiran, an intrathecally administered siRNA targeting APP, is being evaluated in Phase 2 for cerebral amyloid angiopathy ^[7]. Updated Phase 1 data showed durable reductions in CSF biomarkers in participants with up to 30 months of treatment exposure ^[7].

This approach builds on extensive clinical experience with RNAi, but delivery performance still needs to be established for each tissue and route of administration. Cellular uptake, intracellular trafficking and endosomal escape remain important determinants of productive silencing.

City Therapeutics' RNAi Platform: cityRNA Triggers and Delivery Ligands

City places particular emphasis on RNAi trigger engineering. Its three lead liver-directed programs use GalNAc-conjugated siRNAs, while its next-generation cityRNA platform is being developed for additional applications. The disclosed results span human and non-human primate studies ^[8]:

• CITY-FXI (factor XI, antithrombotic): mean FXI reduction of 61 / 79 / 85% at 15 / 50 / 150 mg single subcutaneous doses in Phase 1 Part A.
• CITY-RBP4 (hepatic RBP4, Stargardt disease): about 90% reduction of RBP4 and serum vitamin A in non-human primates.
• CITY-TFR2 (TFR2, anemia of chronic disease): 89% mRNA knockdown in NHP after two doses.

City Therapeutics describes its proprietary cityRNA triggers as approximately 30% shorter and less negatively charged than conventional siRNAs. The company reports that these triggers can productively engage both AGO2 and AGO3 and pairs them with LRP1-targeting ligands to pursue delivery to selected extrahepatic tissues ^[8].

Mechanistic studies show that certain short guide RNAs can activate AGO3-mediated target cleavage, supporting the rationale for alternative RNAi trigger designs ^[9]. However, these findings alone do not establish improved tissue distribution, endosomal escape, or therapeutic activity. Those benefits require validation for each candidate and delivery system.

Figure 3. Mechanistic findings from experimental cityRNA systems of cleaving three different targets by cityRISC ^[9].

Two Areas of Innovation in RNAi

RNAi innovation is advancing through both trigger engineering and tissue-targeted delivery. Alnylam brings extensive clinical experience with chemically modified siRNAs and is developing conjugate strategies to extend delivery beyond the liver ^[5]. City Therapeutics is developing differentiated RNAi triggers alongside delivery strategies designed to reach selected extrahepatic tissues ^[9].

Table 1. Comparison of RNAi platforms, delivery strategies, and clinical development status between Alnylam and City Therapeutics.

Both approaches ultimately require evidence of productive delivery, target-specific gene silencing, and relevant pharmacodynamic activity. Humanized target models can help address sequence-dependent limitations in preclinical evaluation and represent one component of a broader validation strategy ^[11].

Delivery and Target Sequence Challenges

Clinical siRNAs target human transcripts, but their binding sites may differ in mice, particularly in less-conserved regions such as the 3′UTR. Even a guide perfectly matched to human mRNA may show little or no activity against the mouse counterpart ^[11].

This is why so much RNAi efficacy work relies on non-human primates, as the City and Alnylam disclosures show ^[8]. NHP studies are slow, costly and ethically constrained, and they are a poor fit for the iterative screening that modern sequence-optimized chemistry demands ^[11].

When an siRNA or ASO candidate does not adequately recognize the corresponding mouse target sequence because of sequence differences, a humanized target mouse model may provide a more relevant system for evaluating target engagement and pharmacodynamic activity. The suitability of such models depends on the humanized sequence, target transcript expression, tissue distribution, and underlying disease biology. Humanization can address selected species-related limitations.

Humanized Mouse Models for siRNA Pharmacology and PK/PD Studies

The most direct fix is a mouse that carries the human target sequence itself, which is the design principle behind Cyagen's HUGO-GT™ humanized mice. Each line replaces the mouse gene with its full-length human ortholog, and a large fraction of the catalog also humanizes the 5' and 3' UTRs while retaining the native mouse promoter and regulatory context. For an oligonucleotide program that detail matters more than it sounds: a 3'UTR-targeting siRNA then meets, in the animal, the same human sequence it will meet in a patient, expressed under endogenous control. Broad UTR humanization is not standard practice across humanized-mouse catalogs, and it is where these lines go beyond the usual coding-only humanization.

Figure 4. Comparison of transgenic, CDS, partial, and HUGO-GT™ full-length gene humanization strategies, including wild-type and point-mutated human gene replacement.

That makes HUGO-GT™ a natural fit for CRO-run efficacy and PK/PD studies. A humanized line supports long-term dosing, repeat serum and tissue sampling, and durability readouts over the months that a quarterly or twice-yearly siRNA requires. Because the models are stable germline strains, they also support the kind of repeated, comparative screening that single-animal primate work cannot.

AAV-Based Human Target Expression for Early siRNA Screening

For earlier, faster triage, a complementary route is human target delivery, using AAV or non-AAV approaches depending on the organ. Delivering a human target, or a target-plus-reporter cassette, lets a team stand up a humanized readout in weeks and screen many siRNA candidates against the human sequence before committing to a germline model. This approach screens the candidates; the UTR-humanized line confirms the winners in a durable efficacy model. Both slot cleanly into a CRO workflow.

Figure 5. AAV-based siRNA screening using a blood-based luciferase readout.

The financing wave is pricing a move beyond the liver, but each of those programs still has to demonstrate human-sequence knockdown in a living animal. The models that carry the human target, UTRs included, are where that evidence is generated rather than assumed.

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References

[1] City Therapeutics, Inc. City Therapeutics announces closing of $230.0 million upsized initial public offering, including full exercise of underwriters’ option to purchase additional shares [Internet]. 2026 Oct 19 [cited 2026 Oct 22].

[2] Waldron J. ADARx scores upsized $446M IPO, while fellow RNA biotech City maps out Nasdaq plans [Internet]. Fierce Biotech; 2026 Sep 25 [cited 2026 Oct 22].

[3] Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel'in AV, Milstein S, Taneja N, O'Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME, Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier MA, Rajeev KG, Manoharan M. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc. 2014 Dec 10;136(49):16958-61. doi: 10.1021/ja505986a. Epub 2014 Dec 1. PMID: 25434769.

[4] Alnylam Pharmaceuticals, Inc. Alnylam Pharmaceuticals reports second quarter 2026 financial results and highlights recent period progress [Internet]. 2026 Jul 30 [cited 2026 Oct 22].

[5] Brown KM, Nair JK, Janas MM, Anglero-Rodriguez YI, Dang LTH, Peng H, Theile CS, Castellanos-Rizaldos E, Brown C, Foster D, Kurz J, Allen J, Maganti R, Li J, Matsuda S, Stricos M, Chickering T, Jung M, Wassarman K, Rollins J, Woods L, Kelin A, Guenther DC, Mobley MW, Petrulis J, McDougall R, Racie T, Bombardier J, Cha D, Agarwal S, Johnson L, Jiang Y, Lentini S, Gilbert J, Nguyen T, Chigas S, LeBlanc S, Poreci U, Kasper A, Rogers AB, Chong S, Davis W, Sutherland JE, Castoreno A, Milstein S, Schlegel MK, Zlatev I, Charisse K, Keating M, Manoharan M, Fitzgerald K, Wu JT, Maier MA, Jadhav V. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat Biotechnol. 2022 Oct;40(10):1500-1508. doi: 10.1038/s41587-022-01334-x. Epub 2022 Jun 2. PMID: 35654979.

[6] Alnylam Pharmaceuticals, Inc. Delivery platforms [Internet]. [cited 2026 Oct 22].

[7] Cohen S, Ducharme S, Brosch JR, Vijverberg EGB, Sostelly A, Goteti S, et al. Single ascending dose results from an ongoing Phase 1 study of mivelsiran (ALN-APP), the first investigational RNA interference therapeutic targeting amyloid precursor protein for Alzheimer’s disease. Alzheimers Dement. 2025 Jan 9;20(Suppl 6):e084521. doi:10.1002/alz.084521.

[8] City Therapeutics, Inc. Form S-1 registration statement [Internet]. Washington (DC): U.S. Securities and Exchange Commission; 2026 Sep 24 [cited 2026 Oct 22]. Accession No.: 0001104659-26-110430.

[9] Zhang H, Sim G, Kehling AC, Adhav VA, Savidge A, Pastore B, Tang W, Nakanishi K. Target cleavage and gene silencing by Argonautes with cityRNAs. Cell Rep. 2024 Oct 22;43(10):114806. doi: 10.1016/j.celrep.2024.114806. Epub 2024 Oct 4. PMID: 39368090; PMCID: PMC11533134.

[10] Guy R. City: taking RNAi beyond the liver [Internet]. BioCentury; 2024 Dec 20 [cited 2026 Oct 22].

[11] Oliver PL, Hill AC. Model selection in preclinical nucleic acid therapeutics research. Commun Biol. 2026 Feb 9;9(1):200. doi: 10.1038/s42003-026-09650-7. PMID: 41663519; PMCID: PMC12886855.