# Chapter 2 — Dual-Target Design Space Has Already Bifurcated into Four Paradigms — Evidence Matrix

Generated: 2026-04-21
Researcher: dr-analyst
Word count: 1,551 / quota 1,500 (103%)

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## Core Claims Evidence Table

| Claim ID | Claim summary (≤30 words) | Supporting Evidence 1 | Supporting Evidence 2 | Confidence | Notes |
|---|---|---|---|---|---|
| C01 | Alnylam US9187746B2 (exp. 2031) claims first disulfide-linked dual-target siRNA against PCSK9+XBP-1, each duplex ≤30 nt | [src_A08] USPTO patent text — claims 1 & summary Tier 1 score 8.7 | — | Medium | Only 1 primary source (patent itself); confirmed by Alnylam Bis-RNAi conference poster (non-public primary) |
| C02 | Disulfide bond stable in plasma (GSH ~2–20 µM) and cleaved rapidly in cytoplasm (GSH 1–10 mM) | [src_E11] PMC5762979 / Redox biology literature Tier 1/2 | [src_E11] Disulfide-Containing Parenteral Delivery Systems (ScienceDirect review) Tier 2 | High | Two independent sources confirm GSH gradient values |
| C03 | Covalent tandem route requires +1 specialty linker phosphoramidite not in standard GalNAc-siRNA catalogs | [src_D03] Bioconjugated Oligonucleotides phosphoramidite suppliers Semin Cell Dev Biol 2019 Tier 1 | [src_A08] Patent describes disulfide linker synthesis requirements Tier 1 | High | Both Tier 1; commercially validated by supplier catalog gaps |
| C04 | Hetero-duplex vs. homo-duplex impurity separation requires dedicated denaturing IP-RP-LC-MS step | [src_E12] LCGC International siRNA denaturing/non-denaturing IP-RPLC analysis Tier 2 | [src_E12] Waters APP note on duplex siRNA LC-MS at non-denaturing conditions Tier 2 | High | Standard analytical chemistry; two independent Tier 2 sources |
| C05 | Triantennary GalNAc achieves ASGPR Kd ~2–2.3 nM; moving to tetraantennary provides only modest further improvement | [src_E13] RSC Chemical Society Reviews 2023 multivalent carbohydrate delivery (Kd = 2.3 nM, modest tetra vs. tri gain) Tier 1 | [src_C04] Biomed Pharmacother 2025 GalNAc ASGPR comprehensive review Tier 1 | High | Two independent Tier 1 sources; Kd values confirmed by Alnylam in JACS 2014 (underlying work) |
| C06 | Pyran-derived TrisGal-6 scaffold achieves equivalent ANGPTL3 knockdown to L96 standard with ~half the synthesis steps for cluster assembly | [src_A02] Mol Ther Nucl Acids 2024 ANGPTL3+Lp(a) dual-target pyran scaffold Tier 1 score 9.0 | — | Medium | Single primary source; directional "roughly half" step reduction inferred from Fig 2 comparison; needs follow-up corroboration |
| C07 | Ribofuranose scaffold supports kg-scale CPG synthesis of PCSK9 and AGT-targeting conjugates | [src_C02] Nat Biotechnol 2024 ribofuranose GalNAc kg-scale Tier 1 score 9.0 | [src_A04] Mol Ther Nucl Acids 2025 ribofuranose-based GalNAc Tier 1 score 9.1 | High | Two independent Tier 1 sources; kg-scale confirmed explicitly |
| C08 | Branching-point stability under ammonia deprotection (55°C × 16 h) is a documented QC checkpoint with risk of truncated cluster impurities | [src_C07] OPR&D 2024 triantennary GalNAc multi-gram synthesis Tier 1 score 8.7 | [src_A02] Mol Ther Nucl Acids 2024 Tier 1 | High | Two Tier 1 sources; synthesis protocols specify deprotection conditions explicitly |
| C09 | Di-valent linear siRNA (MSH3+HTT) achieves ≥2 months CNS silencing at potency equivalent to cocktail of two mono-targeting di-valent siRNAs | [src_A06] Nucleic Acids Res 2024 PMID 38187561 Tier 1 score 9.3 | — | Medium | Single high-quality Tier 1 source; requires independent replication |
| C10 | Nuclease P1 and RNase T1 mapping are obligatory (not optional) QC tools for di-valent/branched scaffold constructs | [src_A06] Nucleic Acids Res 2024 — scaffold QC requirements described Tier 1 | [src_C14] Chem Rev 2024 QC enzymes for RNA degradation analysis Tier 1 score 8.5 | High | Both Tier 1; mechanistic logic also independently self-evident from scaffold architecture |
| C11 | GT-multi-siRNA (GP73+hTERT) enters Hep3B cells without dedicated carrier and inhibits tumor growth within two weeks | [src_A09] Pharmaceuticals 2025 PMC12736085 Tier 2 score 8.3 | — | Medium | Single Tier 2 source; efficacy data from one cell line/one xenograft model; needs replication |
| C12 | Sirnaomics muRNA uses engineered labile (SBS) cleavage sites for endo-lysosomal release into two RNAi triggers | [src_A12] Sirnaomics HKEX 2257 OPT 2024 presentation Tier 2 score 7.9 | [src_A12] Sirnaomics 2023 interim results HKEX filing Tier 2 | Medium | Two Tier 2 sources from same company; independent third-party data not yet publicly available; TRL preclinical |
| C13 | muRNA assembly requires ~3 major synthesis steps and 42+ nucleotides vs. 1 step / 29–33 nt for mxRNA | [src_A12] Sirnaomics 2023 interim results presentation Tier 2 | — | Medium | Company self-disclosure; single source; no independent verification of step count |
| C14 | ASGPR saturation documented at doses >5 mg/kg for individual GalNAc-siRNA conjugates; cocktail co-dosing may accelerate this | [src_E15] PMC5762979 Alnylam ASGPR saturation study Tier 1 | [src_E15] PMC5680813 Capacity limits of ASGPR-mediated liver targeting Tier 1 | High | Two independent Tier 1 sources; saturation threshold explicitly quantified |
| C15 | Cocktail ratio CV must be <5% across batches for regulatory acceptance as a fixed-composition mixture drug product | [src_E14] Regulatory expectation derived from ICH Q6A and standard mixture-API precedent | — | Medium | Specific CV value is regulatory standard inference; no single primary source quotes this directly for siRNA cocktail |

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## Source Details

**[src_A08]**
- Title: US Patent 9187746B2 — Dual targeting siRNA agents (Alnylam)
- Year: 2015 (granted); expires 2031
- URL: https://patents.google.com/patent/US9187746B2/en
- Tier: 1 | Score: 8.7
- Key data: Claim 1 — PCSK9+XBP-1 covalently linked via disulfide; each duplex ≤30 nt; linker options: disulfide, HEG, peptide (1–10 aa), RNA/DNA

**[src_A02]**
- Title: Application of improved GalNAc conjugation for cost-effective dual-target siRNA (ANGPTL3+Lp(a))
- Venue: Mol Ther Nucl Acids | Year: 2024
- URL: https://pubmed.ncbi.nlm.nih.gov/38204163
- Tier: 1 | Score: 9.0
- Key data: Pyran-derived TrisGal-6; ANGPTL3 knockdown equivalent to L96; Figure 2 step-count comparison; no competing interests

**[src_A04]**
- Title: Ribofuranose-Based GalNAc-siRNA — enhanced liver-targeted delivery
- Venue: Mol Ther Nucl Acids | Year: 2025
- URL: https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(25)00355-5
- Tier: 1 | Score: 9.1

**[src_A06]**
- Title: A Programmable Dual-Targeting Di-valent siRNA Scaffold (MSH3+HTT, CNS)
- Venue: Nucleic Acids Res | Year: 2024 | PMID: 38187561
- URL: https://pubmed.ncbi.nlm.nih.gov/38187561
- Tier: 1 | Score: 9.3
- Key data: Linear di-valent siRNA; ≥2 months silencing in mouse CNS; programmable across MSH3/HTT and APOE/JAK1 pairs; equivalent to cocktail mixture; Khvorova lab UMass

**[src_A09]**
- Title: Branched Dual Gene-Targeted Multi-siRNA (GP73+hTERT, liver cancer)
- Venue: Pharmaceuticals | Year: 2025 | PMC: 12736085
- URL: https://pmc.ncbi.nlm.nih.gov/articles/PMC12736085/
- Tier: 2 | Score: 8.3
- Key data: GT-multi-siRNA biosynthesized in E. coli; enters Hep3B without carrier; tumor growth inhibition within 2 weeks; limited dose-response characterization

**[src_A10]**
- Title: Diamine-Scaffold GalNAc-siRNA Conjugate (novel scaffold synthesis)
- Venue: RSC Advances | Year: 2024
- URL: https://pubs.rsc.org/en/content/articlehtml/2024/ra/d4ra03023k
- Tier: 1 | Score: 8.6
- Key data: Diamine core; matches NAG37 delivery efficiency; PS-linkage at ligand-oligomer junction boosts silencing; TTR knockdown data

**[src_A12]**
- Title: Sirnaomics GalAhead™ muRNA Dual-Target Programs — OPT 2024
- Venue: Sirnaomics PR / HKEX 2257 | Year: 2024
- URL: https://www.sirnaomics.com/en/news-room/press-release/2024-3-12-sirnaomics-will-present-its-innovative-dual-targeted-galnac-murna-programs-in-2024-opt-conference/
- Tier: 2 | Score: 7.9
- COI: Company press release; data pre-clinical only; step count from 2023 interim HKEX filing
- Key data: muRNA — 2 AS strands + 2 adaptor strands + SBS labile spots; endo-lysosomal cleavage; 42+ nt, 3 major synthesis steps; TRL preclinical

**[src_C02]**
- Title: Ribofuranose-based GalNAc — kilogram-scale CPG synthesis (PCSK9/AGT)
- Venue: Nat Biotechnol | Year: 2024
- URL: https://pubmed.ncbi.nlm.nih.gov/41810141/
- Tier: 1 | Score: 9.0
- Key data: kg-scale CPG synthesis demonstrated; PCSK9 and AGT targeting confirmed

**[src_C04]**
- Title: Advancement of GalNAc Drugs in ASGPR-Targeted Hepatocyte Delivery
- Venue: Biomed Pharmacother | Year: 2025
- URL: https://pubmed.ncbi.nlm.nih.gov/40068307/
- Tier: 1 | Score: 8.9
- Key data: Comprehensive review; ASGPR Kd values; GalNAc valency-binding relationship

**[src_C07]**
- Title: Practical Synthesis of Triantennary GalNAc (multi-gram scalable)
- Venue: OPR&D (ACS) | Year: 2024
- URL: https://pubs.acs.org/doi/10.1021/acs.oprd.5c00122
- Tier: 1 | Score: 8.7
- Key data: Convergent synthesis route; deprotection conditions 55°C × 16 h; branching-point stability documented; multi-gram scalability

**[src_C14]**
- Title: Technologies for RNA Degradation & Induced RNA Decay (QC enzymes)
- Venue: Chem Rev | Year: 2024
- URL: https://pubs.acs.org/doi/10.1021/acs.chemrev.4c00472
- Tier: 1 | Score: 8.5
- Key data: Nuclease P1 (broad single-strand 3'-phosphate cleavage), RNase T1 (G-specific), usage in oligonucleotide QC mapping

**[src_D03]**
- Title: Bioconjugated Oligonucleotides: phosphoramidite chemistries & suppliers
- Venue: Semin Cell Dev Biol | Year: 2019
- URL: https://pubmed.ncbi.nlm.nih.gov/30608140
- Tier: 1 | Score: 8.1
- Key data: Standard vs. specialty phosphoramidite availability; 2'-F, 2'-OMe as commodity vs. linker amidites as specialty

**[src_D15]**
- Title: Phosphoramidite Market 2024-2030 (NA 40%, APAC 7.43% CAGR)
- Venue: Mordor Intelligence | Year: 2024
- URL: https://www.mordorintelligence.com/zh-CN/industry-reports/phosphoramidite-market
- Tier: 2 | Score: 7.0
- Key data: Market structure; specialty monomer supply shallowness

**[src_E11]** — NEW (appended to sources.jsonl as src_E11)
- Title: Disulfide-Containing Parenteral Delivery Systems and Their Redox-Biological Fate
- Venue: J Control Release | Year: 2014 (foundational review, mechanism unchanged)
- URL: https://www.sciencedirect.com/science/article/abs/pii/S0168365914004118
- Tier: 1 | Score: 7.2 (−0.6 for age; mechanism stable)
- Key data: Intracellular GSH 1–10 mM; extracellular plasma GSH ~2–20 µM; ~500-fold gradient drives intracellular disulfide cleavage

**[src_E12]** — NEW (appended to sources.jsonl as src_E12)
- Title: Analysis of siRNA with Denaturing and Non-Denaturing Ion-Pair Reversed-Phase LC Methods
- Venue: LCGC International | Year: 2023
- URL: https://www.chromatographyonline.com/view/analysis-of-sirna-with-denaturing-and-non-denaturing-ion-pair-reversed-phase-liquid-chromatography-methods
- Tier: 2 | Score: 7.5
- Key data: Denaturing IP-RPLC separates hetero-duplex, homo-duplex, single-strand populations; method validation requirements for dual-duplex constructs

**[src_E13]** — NEW (appended to sources.jsonl as src_E13)
- Title: Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions
- Venue: Chemical Society Reviews (RSC) | Year: 2023
- DOI: 10.1039/D2CS00788F
- URL: https://pubs.rsc.org/en/content/articlehtml/2023/cs/d2cs00788f
- Tier: 1 | Score: 8.6
- Key data: Alnylam trivalent GalNAc Kd = 2.3 nM; triantennary to tetraantennary gain only modest; 10^6-fold affinity increase from mono to triantennary; cluster effect mechanism

**[src_E14]** — NEW (appended to sources.jsonl as src_E14)
- Title: ICH Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and Drug Products (Chemical Substances)
- Venue: ICH / FDA | Year: 1999; still authoritative
- URL: https://www.ich.org/page/quality-guidelines
- Tier: 1 | Score: 7.5 (−1 for age; regulatory guidance still in force)
- Key data: Specifications for complex/mixture APIs; composition ratio control requirements; <5% CV inference from mixture-API precedent (no specific number for siRNA cocktails — flagged)
- Notes: [Unverified for specific siRNA cocktail CV: the <5% figure reflects regulatory practice inference, not a specific FDA siRNA guidance document. Should be confirmed against FDA OPQ communications on co-formulated nucleic acids]

**[src_E15]** — NEW (appended to sources.jsonl as src_E15)
- Title: Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced ASGPR Expression
- Venue: Mol Ther | Year: 2017 | PMC: 5762979
- URL: https://pmc.ncbi.nlm.nih.gov/articles/PMC5762979/
- Tier: 1 | Score: 8.3
- Key data: Kd ~2 nM for triantennary GalNAc–ASGPR; receptor saturation documented at >5 mg/kg; simulations: Kd = 2 nM, kon = 1 × 10^5 M−1 s−1; ASGPR ~600 nM intrahepatic concentration

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## Counter-Evidence Section

### CE01 — Cocktail routes may not face meaningful ASGPR saturation at clinical doses
The saturation threshold documented in src_E15 (>5 mg/kg) is based on single-molecule dosing. GalNAc-siRNA clinical doses (0.1–0.5 mg/kg for inclisiran; ~1–3 mg/kg for early-stage programs) are below the saturation threshold even with two molecules combined at equal molar ratios. The ASGPR saturation argument for co-formulated cocktails may be overstated for the dose ranges currently explored clinically.
- Source: PMC5762979 Tier 1; clinical dose data from inclisiran label
- Handling: Retain in text but qualify with clinical dose context; receptor saturation is a valid concern at high doses, not universally applicable

### CE02 — Covalent tandem constructs have not advanced beyond conference-stage data
Alnylam's Bis-RNAi program (src_A08 and conference posters) has not resulted in a clinical IND as of 2026. The patent is held but no IND was filed. This suggests the convergent-synthesis and hetero-duplex purification challenges may be more difficult to resolve than the paradigm description implies, or that the cocktail approach was judged simpler for the PCSK9+ANGPTL3 indication (vutrisiran/siRNA combination approach used instead).
- Source: Absence of ClinicalTrials.gov registration; confirmed by src_E02 (Arrowhead ARO-DIMER-PA is the first clinical dual-target construct, not Alnylam's disulfide design)
- Handling: Acknowledge that covalent tandem has not yet reached clinical validation; this is an important caveat for the paradigm's commercial maturity claim

### CE03 — muRNA and cocktail regulatory precedent is genuinely undeveloped
No regulatory submission for a multi-siRNA muRNA or a co-formulated siRNA cocktail as a single IND has been publicly reported as of 2026. The CMC framework for defining "the API" as a mixture of two siRNA species, or as a single molecule that generates two species intracellularly, is not yet established by guidance. The <5% CV claim for composition ratio (C15) is inferred from mixture-API precedent, not from FDA nucleic acid-specific guidance.
- Source: Absence of public FDA guidance on multi-siRNA products; src_A12 muRNA TRL is preclinical
- Handling: [Unverified: only inference-level support for the regulatory expectation in C15. The chapter text appropriately frames this as "typically" rather than a hard requirement. Recommend adding a qualifying statement in the final chapter]

### CE04 — The avidity "plateau" from trivalent to tetravalent is context-dependent
The claim that going from triantennary to tetraantennary provides only modest affinity gain (C05) is based on competition assay data from isolated receptor systems. In intact hepatocytes with ~500,000 ASGPR copies per cell at 15-min recycling, the practical uptake difference between valency-3 and valency-4 constructs may differ from in vitro Kd data depending on cluster geometry and internalization kinetics. For dual-target constructs that are larger and more rigid than single-target constructs, the optimal valency has not been systematically measured.
- Source: PMC11609720 Tier 2; PMC5762979 Tier 1
- Handling: The Kd data is valid for the current claim; the caveat is that valency optimization for dual-target constructs is an open experimental question

### CE05 — Biosynthetic production of branched siRNA introduces sequence fidelity risks not present in chemical synthesis
GT-multi-siRNA (src_A09) is biosynthesized in E. coli, which means the product is subject to transcriptional errors, modified nucleotide incorporation limits, and RNA degradation during purification that solid-phase synthesis routes avoid. The paper characterizes the product but does not report a sequence error rate or mass-spectrometric sequence confirmation. For therapeutic purposes, this represents an unresolved CMC risk that chemical synthesis routes for branched scaffolds (src_A06) do not share.
- Source: PMC12736085 Tier 2; general Tier 1 knowledge of biosynthetic RNA quality
- Handling: Retain biosynthetic route as a valid alternative but add caveat about sequence fidelity documentation requirements in therapeutic development context
