LvNP Gene Editing Delivery Service Platform

1. Concept: Redefining CRISPR Delivery with "Virus-Level Efficiency × Protein-Level Safety"

LvNP (Lentiviral Nanoparticle) is a next-generation gene editing platform that precisely delivers Cas9 RNP in the form of virus-like nanoparticles. It combines the outstanding cell-entry efficiency of lentiviruses with the high safety profile of protein delivery, enabling a highly efficient, transient, integration-free, and DNA-free mode of gene editing delivery. Its core advantages are:

• Safer than lentiviruses: no DNA, no integration, no sustained expression

• More convenient than electroporation: no equipment required, gentler on cells

• More efficient than LNP: broader cell-type applicability, higher editing efficiency

• More precise than plasmids: transient editing, narrow activity window, low off-target risk

Fig1. Schematic diagram of LvNP and product characteristics

2. Technical Principle

LvNP achieves its function throughengineered reconstruction of the HIV-1 Gag nucleocapsid structure, enabling itto:

·      Specifically bind the Cas9–gRNARNP through a targeted recognition system

·      Efficiently load matureCas9–gRNA RNP into virus-like particles

·      Enter target cells via a viralentry mechanism after particle maturation

·      Achieve precise editing withoutDNA templates and without integration risk

This mechanism ensures:

·      Enrichment of "functionalRNP" rather than unbound Cas9

·      High particle purity and strongactivity

·      Efficient, stable, andcontrollable delivery

3. Application Scenarios

3.1 Cell Therapy

LvNP can co-deliver RNP + functional mRNA and is highly suited for immune cell engineering:

• CAR-T cell construction: delivery of CAR mRNA + CRISPR RNP (e.g., TRAC KO)

• TCR-T cell construction: introduction of TCR sequences and knockout of endogenous TCR via RNP

• NK cell engineering: enhanced cytotoxicity, improved metabolism, and increased persistence

3.2 Gene Therapy

LvNP combines efficient delivery, low integration risk, and controllable dosing, making it an ideal gene therapy tool:

• Genetic disease gene correction (ex vivo HSC/iPSC editing; in vivo local delivery)

• Cancer gene therapy (modulation of the immune microenvironment)

• Gene repair and disease model construction

3.3 Stem Cell Research and Engineering

LvNP is particularly well-suited for hard-to-transfect cells such as iPSCs and hematopoietic stem cells (HSCs):

• Stem cell genetic modification and gene correction

• Complex editing of iPSCs (multi-gene knockout/knock-in)

• Stem cell therapy model development

3.4 Gene Editing and Gene Function Research

LvNP is widely used for research-end gene manipulation, leveraging its "integration-free + high-efficiency" profile, including:

• Gene knockout / knock-in / point mutation studies: precise Cas9 RNP delivery for rapid gene editing

• Gene function validation: suitable for cell development, disease modeling, and regulatory mechanism research

• Epigenetics and pathway dissection: transient editing reduces background expression interference, facilitating clearer causal inference

4. Core Technical Advantages: High Efficiency · Transient Editing · Superior Safety · Low Immunostimulation

4.1 Virus-Level Delivery Efficiency Across Multiple Hard-to-Transduce Cell Types

LvNP retains the natural cell-entry advantages of lentiviruses, demonstrating industry-leading transduction performance in:

• Primary cells

• T cells / NK cells

• Stem cells including iPSCs and HSCs

• Neurons and neural progenitor cells

• Adherent and suspension hard-to-transfect cell lines

4.2 Transient Editing — High Precision, Low Off-Target Risk

Cas9 RNP does not require transcription or translation within the cell. It initiates editing immediately upon entry, with a narrow editing window and low background noise:

• Lower off-target risk

• Cleaner, more interpretable editing outcomes

• Higher gene knockout / knock-in efficiency

4.3 Zero Integration, Zero Persistent Expression — Highest Biosafety Standard

LvNP carries no genome, generates no integration events, and represents a truly DNA-free delivery modality:

• Does not enter the host genome

• Does not sustain Cas9 expression

• Does not introduce a potential mutational burden

4.4 Lower Immunostimulation — Suitable for In Vivo Delivery and Long-Term Studies

Compared with other viral or lipid-based delivery approaches:

• Significantly lower immunogenicity

• Compatible with in vivo models

• Applicable to projects with high requirements for "immune safety"

4.5 Broad Compatibility

Supports multiple gene editing tools:

• Cas9 variants (SpCas9, SaCas9, etc.)

• Cas12a / Cas13 family

• Combined delivery strategies for mRNA + RNP

5. Three-Way Comparison: LvNP vs.Lentivirus vs. LNP

5.1 Overview of Basic Characteristics

The three delivery modalities exhibitsystematic differences in vector nature, packaging mechanism, safety, andefficiency.

Table 1. Comprehensive Multi-Dimensional Comparison ofThree CRISPR Delivery Methods

5.2 In-Depth Comparison of Packaging Principles

Lentivirus(LV) Packaging Mechanism: Gag protein recognizesthe ψ packaging signal on viral RNA to encapsulate the RNA genome, with reversetranscriptase co-packaged. After cell entry, the RNA is reverse-transcribedinto cDNA, and integrase embeds the cDNA into the host chromosome, creatingpermanent genetic alterations. The core safety concerns are insertionalmutagenesis risk and persistent expression of the target gene.

LNPPackaging Mechanism: Non-specific encapsulation isachieved through electrostatic self-assembly between ionizable lipids andnegatively charged nucleic acids (mRNA or plasmid). Upon cell entry, cargorelease depends on endosomal escape (efficiency only ~1–2%), after which mRNAis translated into Cas9 protein in the cytoplasm and subsequently assembleswith sgRNA into RNP. Cas9 expression can persist for several days, resulting ina broad off-target window; overall损耗 is substantial; and there is a naturalliver-enrichment tendency.

LvNPPackaging Mechanism: The cargo is the Cas9–sgRNARNP protein complex. Through a dual-specificity mechanism of Gag-fusion + RNAaptamer, RNP assembly and particle encapsulation are completed within producercells. Upon entry into target cells, endosomal escape occurs via the sameenvelope-fusion mechanism as lentivirus. The RNP is directly transported to thenucleus to initiate editing, with no transcription or translation stepsrequired. Cas9 activity lasts only several hours. Integrase inactivation ensuresno residual genetic material.

6. Case Studies

Fig2. LvNP packaging multiple RNPs

Two RNPs targeting VEGFA and ARHGAP32 were codelivered into HEK293T cells, with significant editing at both loci observed within 48  hours.

Fig3. Gene editing efficacy measured at different time points following LvNP packaging and infection

After infecting Jurkat cells with 10 µL of LvNP, editing at the VEGFA target site was assessed at 48 hours. Editing efficiency exceeded 80%, surpassing that achieved by electroporation.

7. Technical Services and Collaboration

We provide a complete LvNP gene editing service system spanning design through validation:

7.1 Custom LvNP Particle Preparation

• RNP packaging for diverse Cas protein / sgRNA combinations

• Support for multiple gene editing tool combinations

• Delivery of high-activity finished LvNP particles

7.2 Cas9/gRNA Project Suite Service

• gRNA target site design

• Off-target prediction and optimization

• Vector construction

• RNP preparation and LvNP packaging

• Cellular editing and phenotypic validation

• Comprehensive analysis and results reporting

• "One-stop gene editing platform service" for enterprises and research institutions.

7.3 Long-Term Collaboration Programs

• Joint R&D

• Co-development projects

• Process development and technology transfer

References

[1] Geilenkeuser J, Armbrust N, Steinmaßl E, et al. Engineered nucleocytosolic vehicles for loading of programmable editors. Cell, 2025, 188: 2637–2655.e2631.

[2] Hamilton JR, Chen E, Perez BS, et al. In vivo human T cell engineering with enveloped delivery vehicles. Nat Biotechnol, 42, 1684–1692 (2024).

[3] Banskota S, Raguram A, Suh S, et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell, 2022, 185(2): 250–265.e16.

[4] Xu D, Besselink S, Ramadoss GN, et al. Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins. Nat Commun, 16, 7948 (2025).