On April 9, 2025, a groundbreaking study published in Cell introduced a novel delivery technology called ENVLPE (Engineered Nucleocytosolic Vehicles for Loading of Programmable Editors), developed by a team of scientists from the Helmholtz Munich Center and the Technical University of Munich. This new system promises to deliver gene editing tools into living cells significantly more efficiently than previous methods, opening doors to more effective genetic therapies and biomedical research.

Figure 1. Screenshot of publication status. (doi: 10.1016/j.cell.2025.03.015)

Gene editing technologies, such as CRISPR-Cas9, base editors, and Prime Editors, have rapidly advanced, providing unprecedented tools for the treatment of genetic diseases and advancements in biomedical research. However, these tools safe and efficient delivery into target cells has been a major bottleneck hindering their clinical translation. Existing delivery systems, such as viral vectors and lipid nanoparticles, have their advantages but are also plagued by issues such as immunogenicity, low delivery efficiency, and off-target risks.

ENVLPE is based on engineered, non-infectious virus-like particles (VLPs), designed to efficiently deliver gene editing tools into living cells. The core breakthrough of this new delivery system is its ability to overcome the limitations of current gene delivery platforms. By using a novel approach to RNP (ribonucleoprotein) complex transport, ENVLPE enhances the delivery of gene editing systems while minimizing undesired off-target effects.

Addressing Challenges in Gene Editing Delivery Systems

Traditional delivery tools, such as Adeno-Associated Viruses (AAV) and lentiviruses, have been widely used but come with their own set of limitations:

• AAVs: Limited payload capacity and difficulty delivering large editing tools (e.g., Prime Editor), as well as concerns over immunogenicity and potential genomic integration risks.
• Lipid Nanoparticles (LNPs): Lack of cell specificity, requiring complex optimization of formulations, and inconsistent delivery efficiency.
• Current VLP systems: Relies on the fusion of Cas proteins with Gag, which results in the delivery of "empty" Cas enzymes not associated with gRNA, increasing off-target risks.

Additionally, Prime Editor's guide RNA (pegRNA) structure, especially the fragile 3' end extension, is susceptible to degradation by nucleases, limiting editing efficiency. Virus-like particles (VLPs), however, offer multiple advantages. VLPs can be designed to target specific cells or tissues by incorporating viral tropism and can deliver gene editing complexes in the form of ribonucleoproteins (RNPs), which eliminates the risk of insertion mutations or activation of host immune responses caused by viral components.

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ENVLPE Design and Mechanisms

The ENVLPE system integrates three major innovations to achieve efficient delivery of gene editing tools:

(1) Nucleocytosolic Shuttling Mechanism: Targeted RNP Complex Transport

• Key Design: A nuclear localization signal (NLS) and nuclear export signal (NES) are inserted into the NC (nucleocapsid) domain of the HIV-1 Gag protein, giving the particle the ability to shuttle between the nucleus and cytoplasm.
• Mechanism: Inside the nucleus, Cas proteins bind with the PP7 aptamer-tagged (pe)gRNA to form RNP complexes. The Gag-PCP protein captures the RNP through the PP7 aptamer and actively transports the RNP from the nucleus to the cytoplasm, where it is packaged into the VLP.
• Advantage: Only fully assembled RNPs are delivered, avoiding the delivery of "empty" Cas enzymes, thus enhancing editing specificity and reducing off-target effects.

Figure 2. The design of ENVLPE.

(2) Csy4-mediated Protection of the 3' End of pegRNA

• Technical Challenge: The 3' extension of pegRNA, which includes the reverse transcription template (RTT) and primer binding site (PBS), is prone to nuclease degradation.
• Solution: The Csy4 protein, sourced from Pseudomonas aeruginosa, binds with high affinity to the C4 aptamer, which is inserted at the 3' end of pegRNA. After Csy4 cleaves the aptamer, it remains bound, creating a physical barrier that protects the RNA from degradation.
• Effect: Prime Editing efficiency is improved by over 10-fold in HEK293T cells and iPSC-derived neurons, with editing accuracy reaching more than 90%.

Figure 3. Csy4-mediated protection of pegRNA.

(3) Modular and Miniaturized Design: From ENVLPE to miniENVLPE

• Modular Adaptability: By swapping the PP7 aptamer on the (pe)gRNA, ENVLPE can be adapted to deliver a variety of CRISPR effectors, including Cas9, base editors, and CRISPR activation systems.
• Miniaturized Version (miniENVLPE): By removing non-essential domains (e.g., MA, CA) from the HIV-1 Gag protein and introducing the GCN4 coiled-coil structure to promote self-assembly, the team developed a "mini" VLP that retains only 13% of the original sequence. miniENVLPE maintains editing efficiency similar to the full version but is smaller and has lower immunogenicity, making it more suitable for clinical applications.

Figure 4. Simplified design of miniENVLPE.

In Vitro and In Vivo Validation: From Cells to Disease Models

(1) In Vitro High-Efficiency Editing

• Prime Editing: In the HEK293T luciferase reporter system, ENVLPE-mediated editing efficiency reached 35%, and 30% in iPSC-derived cortical neurons.
• Base Editing (BE): Targeting the splicing site of the B2M gene in T lymphocytes resulted in 90% editing efficiency, enabling the successful knockout of MHC-I and TCR, offering new strategies for the development of universal CAR-T cells.
• Homology-Directed Repair (HDR): Using integrase-deficient lentiviral (IDLV) delivery of repair templates, HDR efficiency reached 40% in HEK293T cells.

Figure 5. ENVLPE+ optimization for PE and BE at endogenous sites.

(2) In Vivo Therapeutic Validation

In this study, the team tested ENVLPE in a mouse model of hereditary blindness, where the mice carried a mutation in the Rpe65 gene, essential for the production of photoreceptor molecules in the retina, rendering them completely blind. After ENVLPE was injected subretinally to correct the mutation, the mice began to respond to light stimuli. "The extent of recovery was astounding. This indicates that the ENVLPE system holds therapeutic potential in living animals," said Julian Geilenkeuser, one of the co-authors of the paper. Compared to existing systems, ENVLPE showed significantly better results, requiring more than 10 times lower doses to achieve comparable effects.

Figure 6. Subretinal injection of ENVLPE for mRNA and PE-RNP delivery in Cre-reporter and retinal degeneration models.

Conclusion and Future Outlook

This research highlights the superior editing efficiency and therapeutic potential of ENVLPE. Moving forward, the team plans to harness the diversity of nature and AI-assisted protein design to develop more cell- or tissue-specific delivery systems. To bring ENVLPE to clinical applications, the research team is seeking funding from pharmaceutical industry partners for translational support.