Cell therapies represent an innovative approach to treating diseases by reprogramming cells to enhance or restore their function. These therapies commonly use immune cells sourced from either the patient (autologous) or a suitable donor (allogenic). Once isolated, the cells are reprogrammed, expanded, and infused back into the patient, to target specific diseases.

One of the promising technologies driving these advancements is lipid nanoparticles (LNP). LNP offer a flexible non-viral gene delivery platform for delivering ribonucleic acid (RNA) that can modulate gene expression or enable precise gene editing.

Reprogramming cells with RNA-LNP

  1. T cell isolation: The patient’s T cells are isolated.
  2. mRNA encapsulation:  mRNA encoding the chimeric antigen receptor (CAR) that targets disease-specific biomarkers, is encapsulated into LNP.
  3. mRNA delivery: The LNP mediates delivery of the mRNA into T cells ex vivo.
  4. CAR expression: T cells translate the mRNA into CAR proteins, which are presented on the cell surface.
  5. T cell expansion and infusion: The engineered CAR T Cells are expanded and infused back into the patient where they mount an immune response to the target recognized by the CAR.

Fig 1. Autologous chimeric antigen receptor T cells engineering with non-viral gene delivery

This approach leverages RNA encapsulated in LNP, providing a more streamlined and scalable alternative. LNP-based delivery doesn’t require cell culture production, simplifying manufacturing and shortening development timelines.

Cell therapy development

Developing cell therapies is a multi-step process, from selecting the right target to scaling up manufacturing. In cell therapy, the RNA-LNP is used as a reagent, not as the final drug product. However, non-viral gene delivery can enable modulating gene expression and engineering cells into cell therapies and offer several key benefits:

  • Simplified production: LNP do not require complex cell culture systems, making the process faster and more scalable
  • Lower risk of genome integration:  mRNA does not integrate into the host genome, reducing the risk of unintended genetic modifications
  • Versatility: LNP can be easily adapted for use in different cell types and applications, from CAR T cell therapies to gene editing.

Aspects of the framework shown below can help guide and accelerate the optimization of the RNA-LNP ex vivo reprogramming reagent, which is essential for producing the cell therapy product.

Fig 2. Genetic medicine framework that supports the development of cell therapy reprogramming reagents

  1. Target selection: The first step is choosing the antigen, typically biomarkers overexpressed in cancer cells. mRNA is used to encode CAR structures, allowing researchers to rapidly test different CAR designs with LNP as a delivery mechanism. This enables a fast and flexible approach to screening various CAR constructs.
  2. Vector selection: Non-viral methods like mRNA-LNP offer advantages as mRNA presents a lower risk of genome integration and simplifies cell-free manufacturing, making it scalable. The lower risk of mRNA-LNP delivery accelerates the process from target validation to clinical application. Precision tools like CRISPR/Cas9 enable precise editing of DNA in cells. LNP technology can be used to deliver CRISPR components to cells, including T cells, allowing targeted modifications for therapeutic purposes.  A study showed that the Cas9 endonuclease can be encoded in mRNA and co-encapsulated with one or more guide RNA into an LNP and delivered to the liver in vivo (1,2).
  3. Delivery and formulation technology: Cytiva’s GenVoy-ILM™ delivery platform offers a lipid library designed for efficient gene delivery to multiple T cell subsets. These LNP facilitate the delivery of mRNA into different types of T cells without the complications of a packaging cell line, providing a powerful tool for engineering immune cells.
  4. Manufacturing: The NanoAssemblr™ platforms enable scalable production from lab-scale to commercial-scale volumes. Using our proprietary NxGen™ mixing technology, researchers can produce high-quality RNA-LNP reagents for reprogramming cells in a streamlined reproducible process.

Genomic medicine toolkit for cell therapy

Fig 3. Genomic medicine tool kit

Cytiva’s genomic medicine toolkit comprises essential non-viral, lipid-based technologies for gene delivery. From RNA-LNP engineering to our scalable NanoAssemblr™ manufacturing platforms, we offer innovative tools to accelerate the development of next-generation cell therapies. For researchers, our GenVoy-ILM T™ cell kit provides an optimized solution for the delivery of mRNA or CRISPR Cas9 mRNA with gRNA into human primary T cells using LNP prepared on the NanoAssemblr™ Spark™ and NanoAssemblr Ignite™ systems. Our proprietary LNP formulations have been shown to mediate high transgene expression and good cell viability in Primary Human T Cells. Flow cytometry studies demonstrate that certain LNP formulations can achieve high levels of transgene expression without affecting cell viability, a crucial factor in maintaining the yield of modified cells. This approach also offers a distinctive advantage as LNP-based delivery systems are not subject to the same regulatory requirements as traditional drug products, enabling faster and more flexible manufacturing processes. With a robust portfolio of LNP technologies, we are committed to advancing cell therapy development.

References:

  1. Finn JD, Smith AR, Patel MC, et al. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Reports. 2018;22(9):2227-2235. doi:https://doi.org/10.1016/j.celrep.2018.02.014
  2. Miller JB, Zhang S, Kos P, et al. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. Angewandte Chemie International Edition. 2016;56(4):1059-1063. doi:https://doi.org/10.1002/anie.201610209