In Vivo Gene Therapy: Industry Solution

1. Industry Background

In vivo gene therapy (in vivo gene therapy) delivers gene editing tools or therapeutic genes directly to target tissues within the patient, achieving gene knockout, repair, or expression regulation in situ. This approach avoids the complex ex vivo cell manipulation process, offering 'ready-to-use' advantages and broader clinical indication potential.

However, in vivo delivery continues to face three core challenges:

• Insufficient delivery efficiency (particularly in difficult-to-transfect cells and complex tissues)
• Long-term safety risks (viral integration, persistent expression)
• Clinical translation and regulatory pressure

Shutong Medical has built a comprehensive in vivo gene therapy solution centered on the MiniCircle DNA vector and LvNP (Lentivirus Nanoparticle) delivery system, addressing the above pain points while balancing efficiency, safety, and clinical feasibility.

2. Solution Overview

MiniCircle DNA × LvNP Delivery × Full-Spectrum Safety Assessment

Shutong Medical provides a one-stop in vivo gene therapy technology service—from delivery vector preparation and efficient in vivo delivery, to editing efficiency validation and safety assessment—empowering clients to efficiently advance preclinical research and IND filing.

Core Advantages:

• Virus-level delivery efficiency

• Non-integrating safety design

• Highly controllable in vivo expression

• Complete data package meeting regulatory expectations

3. Technical Workflow

Conventional plasmid → MiniCircle conversion → Bacterial backbone removal → High-purity MiniCircle DNA

MiniCircle DNA / mRNA / Editing protein → LvNP engineered assembly → Virus-like nanoparticle formation → Efficient in vivo delivery

LvNP forms virus-like particles through self-assembly of engineered Gag/Gag-Pol structural proteins, efficiently encapsulating functional molecules during particle maturation, and achieving highly efficient delivery to target cells in vivo by leveraging the lentivirus's natural membrane fusion and cell entry mechanisms.

Animal dosing → Multi-tissue sampling → NGS amplicon sequencing → Quantitative editing efficiency analysis

Off-target analysis / Integration site analysis / Chromosomal structural variation detection / Biodistribution detection

4. Module Details

Module 1: MiniCircle DNA

Minicircle DNA (mcDNA) is a non-viral, extrachromosomal, covalently closed supercoiled circular gene expression vector derived from conventional plasmids via site-specific recombination technology, which removes the bacterial backbone sequences (e.g., origin of replication, antibiotic resistance genes, unmethylated CpG motifs, etc.).

Its core feature is retaining only the eukaryotic expression cassette (including promoter, gene of interest (GOI), and polyA signal), while removing prokaryotic sequences that may cause immunogenicity, gene silencing, or integration risks in conventional plasmids. This 'minimalist' design makes MiniCircle DNA a safer and more efficient gene delivery tool.

Module 2: LvNP (Lentivirus Nanoparticle) Delivery System

2.1 What is LvNP?

LvNP (Lentivirus Nanoparticle) is a virus-like nano-delivery system based on the self-assembly of lentiviral structural proteins. Unlike conventional lentiviral vectors, LvNP:

• Does not carry the complete viral RNA genome
• Does not undergo reverse transcription
• Does not integrate into the host genome
• Does not possess replication capacity

The design philosophy is to maximally preserve the lentivirus's naturally efficient cell entry and membrane fusion capabilities, while completely eliminating the safety risks associated with insertional mutagenesis and persistent expression, thereby providing a superior delivery solution for in vivo gene editing and transient expression applications.

2.2 Fundamental Differences Between LvNP and Other Major Delivery Systems

2.3 Engineered Features of LvNP

• Retains only lentiviral structure- and assembly-related functional modules
• Contains no genetic elements related to viral replication or integration
• Capable of efficiently delivering Cas9 RNP, Base Editor protein, Prime Editor complex, and more
• Does not form a persistent expression vector after delivery, significantly reducing long-term safety risks
• Suitable for single-dose or limited-dose in vivo gene editing strategies

2.4 LvNP Delivery Services

2.5 Summary of LvNP Technical Advantages

• High delivery efficiency: Inherits lentivirus's natural cell entry and membrane fusion advantages
• Non-integrating safety: No integration into the host genome
• Highly compatible with gene editing: Especially suited for protein-level delivery such as Cas9 RNP
• Controllable expression: Avoids potential toxicity risks associated with long-term expression
• Strong in vivo compatibility: Applicable across multiple tissues and complex in vivo environments

Module 3: In Vivo Delivery & Efficiency Validation

Scenario 1: Therapeutic Gene Expression Delivery (MiniCircle DNA as Representative)

Core objective: Validate successful delivery, transcription, and protein expression of the therapeutic gene in target tissue.

Detection items:

• Transgene mRNA transcription level: Quantitatively detect mRNA expression abundance of the exogenous gene in target tissue via RT-qPCR or RT-ddPCR, evaluating transcription efficiency and duration.

• Protein expression level: Validate target protein expression intensity and distribution patterns in the target organ using Western Blot, ELISA, or immunohistochemistry (IHC).

• Tissue distribution differential analysis: Compare expression level fluctuations across different doses and time points to provide data support for defining the therapeutic window.

Scenario 2: Gene Editing Delivery (Cas9 mRNA / Base Editor / RNP as Representatives)

Core objective: Quantitatively evaluate the precise editing capability of gene editing tools in living tissues.

Detection items:

• Indel frequency: For CRISPR/Cas9-mediated gene knockout, analyze the insertion/deletion ratio generated by non-homologous end joining (NHEJ) via high-depth NGS amplicon sequencing (>10,000x).

• Base conversion rate: For base editors (Base Editor), calculate the precise conversion frequency of target bases (e.g., C→T or A→G).

• Prime editing efficiency: For Prime Editing, assess the precise insertion, deletion, or substitution efficiency of the target sequence.

• Tissue distribution differential analysis: Compare editing efficiency fluctuations across different doses and time points to provide a basis for clinical dose selection.

Module 4: Off-Target Safety Assessment

This module applies to Scenario 2 (gene editing delivery) and provides a 'Predict + Experiment + Validate' dual-assurance approach for the cleavage risk of nuclease-containing gene editing tools at unintended sites.

Detection strategy (per NMPA Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products (Trial), §8.1.6):

Phase 1: In Silico Prediction

Computational biology methods are used to broadly screen and predict potential off-target sites genome-wide. This phase is the first step in the off-target site identification process, aimed at constructing a theoretical candidate list of potential off-target sites. The core is using algorithmic models to simulate the binding specificity of gene editing tools (e.g., sgRNA) to genomic DNA.

Phase 2: Cell-Based Experimental Simulation

This phase is the second step in off-target site identification—a critical experimental discovery process. It employs high-sensitivity unbiased detection methods in in vitro cell lines that simulate in vivo conditions to comprehensively assess the safety risk profile of gene editing tools. Assessment covers three dimensions: genome-wide unbiased off-target discovery through GUIDE-seq and AID-seq to directly capture all cleavage sites; chromosomal rearrangement detection via PEM-seq to precisely identify translocations, inversions, large-scale deletions, and other structural variations; and karyotyping via G-banding chromosome analysis to detect numerical chromosome abnormalities and large-scale structural variations, systematically assessing genomic stability risk from the molecular to chromosomal level.

Phase 3: In Vivo Targeted Confirmation

Per NMPA Technical Guidelines for Pharmaceutical Research and Evaluation of In Vivo Gene Therapy Products (Trial), §8.1.6: 'For detected off-target sites, risk analysis may be conducted based on site location and gene function; where necessary, animal or human trial data may be integrated for comprehensive judgment.'

In the animal model most closely approximating clinical application, all potential off-target sites identified in the first two phases undergo final targeted validation and quantitative analysis.

Module 5: Integration Site Analysis (ISA)

This module applies to all in vivo gene therapy programs (both Scenario 1 and Scenario 2) and is designed to verify whether the LvNP delivery vector has undergone any unintended integration into the host genome.

• Validating LvNP's non-integrating safety design: LvNP is designed as a non-integrating delivery system that carries no reverse transcriptase and contains no integrase-related genetic elements. This assay experimentally confirms its non-integrating properties, ruling out insertional mutagenesis risk.

Detection Method:

Cell-free DNA (cfDNA) represents DNA fragments released into the blood by cells throughout the body (including cells modified by the vector), providing a 'molecular snapshot' of genome-wide events. Ultra-high-sensitivity detection of vector–genome junction fragments within cfDNA enables non-invasive, comprehensive, and dynamic monitoring of the genome-wide integration profile and clonal evolution in in vivo gene therapy, achieving early warning of potential insertional mutagenesis risks. It can detect potential dominant clonal expansion earlier than clinical symptoms or conventional hematological indicators, providing a valuable time window for clinical intervention and risk management. The core technology is a targeted amplification approach based on Linker-Mediated PCR (LM-PCR), combined with Unique Molecular Identifier (UMI) technology, enabling high-sensitivity, high-specificity detection of viral integration sites in cfDNA. This technology is known as LiBIS-seq (Liquid Biopsy Integration Site Sequencing).

• Individualized risk baseline establishment: After the first administration, a comprehensive individualized integration site risk profile is established for each subject to identify high-risk integration events.
• Long-term dynamic clonal monitoring: During the 15-year long-term follow-up (LTFU) period, periodic cfDNA testing is performed to dynamically track changes in clonal abundance and promptly detect abnormal expansion signals.
• Safety event attribution: Should serious adverse events such as secondary tumors occur, this technology can rapidly identify the integration site of the causative clone, providing definitive evidence for event attribution.
• Regulatory submission support: Provides integration site safety data meeting FDA/NMPA requirements for IND/BLA filings.

Module 6: Biodistribution Detection

Characterizes the pharmacokinetic (PK) profile and tissue tropism of the drug after administration.

• Quantitative PCR (qPCR/ddPCR) monitoring: High-sensitivity detection of vector copy number (VCN) change profiles across different tissues and organs.
• Therapeutic molecule tracking: Concurrent monitoring of MiniCircle DNA transcription levels or mRNA/protein expression kinetics to confirm drug residence time in the target organ.
• Clearance kinetics study: Evaluates the excretion and metabolic kinetics of the delivery system and genetic vector in vivo, confirming that the drug does not produce unintended long-term accumulation in non-target organs.

5. Service Models

Model 1: MiniCircle + LvNP Delivery Package

For clients requiring a complete delivery solution:

• Parental plasmid construction

• MiniCircle production (mg scale)

• LvNP encapsulation and characterization

• In vitro transfection efficiency validation

• Technical consulting support

Suitable for: Early-stage in vivo gene therapy R&D clients

Model 2: In Vivo Safety Assessment Package (IND Filing)

Deliverable: Complete testing report meeting IND filing requirements

Model 3: Individual Services

Select as needed: MiniCircle preparation, LvNP encapsulation, off-target detection, integration site analysis, biodistribution detection

6. Client Value

7. Why Choose GeneRulor

• MiniCircle Preparation: Independently established MiniCircle production platform, capable of providing research-grade to GMP-grade MC plasmids.
• LvNP Delivery Technology: Mature LvNP formulation development and encapsulation processes supporting delivery of multiple nucleic acid types.
• Full-Spectrum Safety Assessment: One-stop coverage of off-target detection, integration site analysis, chromosomal safety assessment, and biodistribution detection.
• Regulatory Compliance Support: Deep familiarity with FDA/NMPA safety requirements for in vivo gene therapy products.