Research & Development

In Vivo CAR Therapy: Innovation and Challenges of Viral Vector Technology

I. Concept and Development History
In Vivo CAR therapy is an innovative technology that disrupts traditional cell therapy models. Unlike conventional CAR-T therapies, which require extracting and modifying a patient’s T-cells ex vivo, In Vivo CAR therapy directly delivers the CAR gene into the patient’s body using delivery systems such as viral vectors or nanoparticles, enabling the gene editing of T-cells in vivo to express Chimeric Antigen Receptors (CARs) that target and kill tumor cells. The advantages of this technology are significant:

1. Shorter treatment cycle: No need for ex vivo cell culture, reducing the treatment time from weeks to just a few days.

2. Lower cost: Simplified production processes, with an estimated cost of only 1/50 to 1/100 that of traditional therapies.

3. Broad applicability: Expected to overcome the challenges of treating solid tumors and reduce immune rejection risks.

Development History:

• Early exploration (2021): U.S.-based Umoja Biopharma used lentiviral vectors to engineer T-cells in vivo, with preclinical results showing CAR-T cells could inhibit tumor growth.

• Technological breakthrough (2022): The team from the University of Pennsylvania successfully used mRNA-LNP (lipid nanoparticle) technology to treat cardiac injury in animal models, proving the feasibility of transient CAR expression in vivo.

• Domestic progress (2023): Beijing BaiTi Biotech (BTBT) developed the "LINCMECAR" technology, which uses nanoparticle vectors to deliver CAR genes and became the first domestic platform to enter preclinical studies.

II. Key Production Processes of Viral Vector Technology

Viral vectors are at the core of In Vivo CAR therapy production. The main technical routes include lentiviral (LV) vectors, adeno-associated virus (AAV) vectors, and mRNA lipid nanoparticle (LNP) delivery systems. The production process involves: gene sequence design → vector construction → quality control → large-scale production. The challenges lie in optimizing vector targeting and maintaining stability during large-scale production.

The production process flow is as follows:

1. Vector Design and Construction: A viral envelope protein targeting T-cells (e.g., CD8-specific fusion protein) is selected. The lentiviral vector genome is designed to include the CAR gene and regulatory elements.

2. Viral Packaging: Using transient transfection, the vector plasmid, packaging plasmid, and envelope plasmid are co-transfected into HEK-293T cells. Transfection reagents like PEIpro® or FectoVIR®-LV are used to increase viral titer.

3. Viral Purification and Concentration: Ultracentrifugation and chromatography techniques are used to remove cell debris and free nucleic acids, ensuring viral purity.

For lentiviral vector-based In Vivo CAR products, the viral titer is a key quality attribute (CQA). Researchers have faced challenges in accurately assessing product quality and optimizing production processes.

The following are commonly used methods for lentiviral titer detection:

For researchers, using RT-qPCR to detect lentiviral titers can more effectively assist in optimizing In Vivo CAR production processes.

To help researchers optimize In Vivo CAR viral titer detection, BlueKit has developed the Lentiviral Vector RNA Copy Number Detection Kit (Catalog No. HG-VR001) for lentiviral titer detection. The product targets the LTR conserved region, making it suitable for all current lentiviral vector backbones. It includes a two-step DNase digestion process to effectively eliminate interference from residual plasmid DNA.

III. Major Research Companies and Technological Comparisons

1. International Companies:

Umoja Biopharma (USA): Uses the VivoVec lentiviral vector platform, which targets T-cells via CD8 envelope protein, and combines with the RACR system to enhance CAR-T cell expansion in vivo. Preclinical data show that a single administration generates functional CAR-T cells in vivo.

Sana Biotechnology (USA): Develops Fusogen technology, which uses fusion proteins targeting T-cell membrane proteins to directly deliver CAR genes to T-cells, avoiding the genomic integration risks associated with viral vectors.

Ixata (UK): Combines lentivirus and LNP technologies, encapsulating lentiviral vectors in nanoparticles to improve in vivo delivery efficiency and reduce immunogenicity.

2. Domestic Companies:

Beijing BaiTi Biotech (BTBT): Developed the "LINCMECAR" platform, using polymer nanoparticles to deliver CAR genes. It has achieved initial success in engineering T-cells to target liver cancer, with costs only 1% of traditional therapies. It is currently in preclinical stages.

Aibo Biotech: Based on mRNA-LNP technology, it explores transient CAR expression in vivo, though its viral vector production still relies on imported lentiviral raw materials.

Fosun Kite and WuXi AppTec: Focus primarily on traditional CAR-T therapies but are gradually localizing viral vector production by introducing overseas technologies (e.g., Fosun Kite’s partnership with Kite Pharma). They plan to achieve domestically produced lentiviral vectors by 2026.

IV. Challenges and Future Outlook

1. Technological Challenges

Targeting and Safety Balance: Viral vectors are prone to being captured by the liver, leading to off-target effects and hepatotoxicity. Engineering the viral capsid or modifying targeting ligands is necessary to improve specificity.

Persistence and Dose Optimization: Lentivirus-mediated CAR expression is long-lasting, which may lead to insertional mutagenesis risks. In contrast, mRNA-LNP has higher safety but requires frequent administration.

2. Industrialization Prospects

Policy Support and Cost Reduction: Starting in 2025, China will implement zero-tariff policies on viral vectors for CAR-T therapies, reducing the cost of imported raw materials by 50%-70%, accelerating domestic process development.

Technological Integration and Innovation: The combination of CRISPR gene editing and viral vectors (such as site-specific integration of CAR genes) could become the next-generation direction, improving safety and expression stability.

V. Conclusion
In Vivo CAR therapy's viral vector technology is leading cell therapy into the “in vivo factory” era. Despite challenges in vector construction, large-scale production, and quality control systems, policy support and the introduction of foreign technologies present opportunities for domestic production. With breakthroughs in targeted delivery systems and automated production processes, In Vivo CAR therapy is poised to become a universal solution for tumor immunotherapy.