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Hydrophobic CPP/HDO Conjugates: A New Frontier for Oligonucleotide-Warheaded PROTAC Delivery

Abstract

Targeted protein degradation has rapidly evolved from small-molecule PROTACs into a broader modality space that now includes nucleic acid–based degraders capable of addressing historically “undruggable” targets such as transcription factors. However, the clinical translation of oligonucleotide-warheaded PROTACs has been fundamentally limited by intracellular delivery barriers. A recent study by Naganuma, Ohoka, Demizu and colleagues introduces a compelling solution: hydrophobic cell-penetrating peptide (CPP)–heteroduplex oligonucleotide (HDO) conjugation, enabling transfection-independent intracellular delivery of decoy oligonucleotide PROTACs and functional degradation of estrogen receptor α (ERα) (Naganuma et al., 2024). This platform represents a major conceptual advance in nucleic acid–based targeted degradation, with broad implications for transcription factor biology, oncology, and nucleic acid drug development.

1. Expanding the Scope of Targeted Protein Degradation

The PROTAC field has matured into one of the most transformative approaches in drug discovery, enabling selective elimination of disease-driving proteins through ubiquitin–proteasome recruitment rather than enzymatic inhibition (Li & Crews, 2022). Classic PROTACs have largely been constrained to proteins with known ligandable pockets, leaving transcription factors and scaffolding proteins largely inaccessible.

Over the last five years, however, nucleic acid–based degraders have emerged as a powerful extension of the paradigm. These include:

  • Decoy oligonucleotide PROTACs (TF-PROTACs) targeting DNA-binding transcription factors (Liu et al., 2021)

  • Aptamer-PROTACs enabling tumor-selective degradation (Liu et al., 2023)

  • Oligonucleotide-PROTACs (O′-PROTACs) incorporating novel cereblon ligands (Yan et al., 2022)

Collectively, these approaches provide a modular route to degrade proteins that lack small-molecule ligands by leveraging their intrinsic DNA-binding specificity (Wang et al., 2022).

Yet one central challenge remains unresolved:

How do we deliver these large, anionic oligonucleotide PROTAC constructs into cells efficiently and safely?

2. Delivery: The Bottleneck for Oligonucleotide-Based PROTACs

Unlike small molecules, oligonucleotide degraders face multiple barriers:

  • High negative charge prevents passive membrane diffusion

  • Susceptibility to nuclease degradation

  • Endosomal trapping after uptake

  • Dependence on lipid transfection reagents in vitro

  • Lack of scalable clinical delivery systems beyond liver-targeting conjugates

Indeed, essentially all previously reported decoy PROTACs required lipofection or nanoparticle formulations, limiting translational potential (Naganuma et al., 2024).

This mirrors broader challenges in oligonucleotide therapeutics, where delivery remains the dominant hurdle despite decades of progress in antisense and siRNA platforms (Hammond et al., 2021).

This is where the new CPP/HDO strategy becomes particularly significant.

3. The CPP/HDO-PROTAC Concept

Naganuma et al. developed a hybrid construct integrating:

  • A 30-mer DNA decoy strand conjugated to an E3 ligase ligand (LCL161 targeting IAP)

  • A complementary strand extended with a 9-mer RNA segment

  • Conjugation of a hydrophobic CPP (P4 peptide: LGAQSNF)

The resulting double-stranded molecule is termed:

CPP/HDO-PROTAC (Naganuma et al., 2024).

A key mechanistic innovation is that the RNA segment creates an RNase H–cleavable site, allowing intracellular release of the active decoy PROTAC after entry.

Conceptually, the construct operates as a delivery-and-activation system: the CPP promotes cellular uptake, while intracellular RNase H processing detaches the peptide and restores the functional oligonucleotide degrader.

3.2 Why Hydrophobic CPPs Matter

Most CPP delivery approaches rely on cationic peptides, which often aggregate with nucleic acids due to electrostatic interactions.

Instead, the authors selected a hydrophobic CPP, originally identified in Duchenne muscular dystrophy antisense delivery screens, avoiding charge-driven aggregation and enabling efficient conjugation chemistry (Jirka et al., 2014; Jirka et al., 2018).

This is an important conceptual shift:

Hydrophobic CPPs may represent a more scalable delivery approach for oligonucleotide-degraders than classical arginine-rich CPPs.

4. Key Experimental Findings

4.1 Binding Affinity Gains

Competitive binding assays revealed striking improvements in ERα affinity. Extended duplex constructs achieved substantially tighter binding than earlier single-stranded decoy PROTACs, likely reflecting additional stabilizing interaction points between ERα and the longer oligonucleotide architecture (Naganuma et al., 2024).

Notably, the CPP-conjugated construct preserved nanomolar binding potency, indicating that peptide attachment does not compromise target engagement.

4.2 RNase H Cleavage Enables Activation

A defining feature of the HDO strategy is its susceptibility to RNase H. The authors demonstrated that the RNA-containing heteroduplex is selectively cleaved, supporting the model that intracellular processing can liberate the active decoy PROTAC once uptake occurs (Naganuma et al., 2024).

This builds directly on the broader HDO concept first established for antisense gene silencing (Nishina et al., 2015).

4.3 Transfection-Independent ERα Degradation

Most notably, CPP/HDO-PROTAC induced degradation of ERα in ERα-positive breast cancer cells without requiring lipid transfection reagents (Naganuma et al., 2024).

This represents a major translational milestone: the first demonstration that a decoy oligonucleotide-based PROTAC can function intracellularly under non-transfection conditions.

4.4 Functional Growth Inhibition

The construct also suppressed proliferation of ERα-positive breast cancer cells over multi-day exposure, consistent with disruption of estrogen signaling pathways (Naganuma et al., 2024).

Given ERα’s established role in breast cancer progression (Kumar & Chambon, 1988), this functional phenotype reinforces the therapeutic relevance of transcription factor degradation strategies.

4.5 Uptake and Endosomal Localization

Fluorescence-based uptake studies showed that CPP conjugation substantially enhances intracellular accumulation. However, most material remained localized within endosomal and lysosomal compartments, highlighting that endosomal escape remains an important barrier for future optimization (Naganuma et al., 2024; Hammond et al., 2021).

5. Broader Context: Where This Fits in the Field

5.1 Nucleic Acid–Based Targeted Degradation is Accelerating

Recent years have seen rapid diversification:

  • TF-PROTACs enabling transcription factor degradation (Liu et al., 2021)

  • O′-PROTACs with cereblon ligands (Yan et al., 2022)

  • Aptamer degraders targeting nucleolin, c-Myc, and mutant p53 (Zhang et al., 2022; Wang et al., 2024; Kong et al., 2024)

The CPP/HDO platform provides a missing piece: delivery without formulation dependence.

5.2 Parallels with Oligonucleotide Therapeutics Delivery

The HDO approach derives directly from antisense delivery advances:

  • DNA/RNA heteroduplexes with enhanced potency (Nishina et al., 2015)

  • Systemic heteroduplex delivery strategies for difficult tissues (Kaburagi et al., 2022)

  • Persistent challenges in intracellular trafficking (Hammond et al., 2021)

By adapting HDO chemistry into PROTAC design, this work bridges two previously separate therapeutic technology stacks (Naganuma et al., 2024).

6. Translational Outlook and Remaining Challenges

Major Opportunities

  • Targeting transcription factors broadly through motif-based decoys (Wang et al., 2022)

  • Eliminating lipid nanoparticle dependency

  • Modular expansion to other E3 ligases and warheads

  • Potential oncology applications beyond ERα

Key Challenges Ahead

  • Improving potency (micromolar activity remains high for clinical relevance)

  • Achieving efficient endosomal escape

  • Enhancing tissue specificity beyond cell culture

  • Stabilizing RNA segments against premature degradation

  • Scaling synthesis and regulatory characterization

As the authors emphasize, DDS optimization will be essential for clinical translation (Naganuma et al., 2024).

Conclusion

The development of hydrophobic CPP/HDO-conjugated PROTACs represents a landmark step toward practical intracellular delivery of oligonucleotide-based targeted degraders. By demonstrating transfection-independent degradation of ERα, this work establishes a new framework for transcription factor degradation, bridging nucleic acid therapeutics and PROTAC engineering (Naganuma et al., 2024).

If successfully extended with improved endosomal escape and tissue targeting, CPP/HDO-PROTACs may unlock an entirely new class of programmable degraders against the most elusive proteins in oncology and beyond.

References (APA Style)

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Kong, L., Meng, F., Zhou, P., Ge, R., Geng, X., Yang, Z., … Ma, J. (2024). An engineered DNA aptamer-based PROTAC for precise therapy of p53-R175H hotspot mutant-driven cancer. Science Bulletin, 69(13), 2122–2135.
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Hydrophobic CPP HDO conjugates …

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Yan, Y., Shao, J., Ding, D., Pan, Y., Tran, P., Yan, W., … Huang, H. (2022). 3-Aminophthalic acid, a new cereblon ligand for targeted protein degradation by O′-PROTAC. Chemical Communications, 58(14), 2383–2386.
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Zhang, L., Li, L., Wang, X., Liu, H., Zhang, Y., Xie, T., … Sun, X. (2022). Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor selective degradation of nucleolin. Molecular Therapy – Nucleic Acids, 30, 66–79.
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