Hit-and-run cell-based therapy research with Trilink mRNA

T cells response against cancer cell

This summer, Moffett et al. published in Nature Communications (Nature Communications – doi:10.1038/s41467-017-00505-8) a new approach for cancer therapeutic research and development based on mRNA delivery. They called it ‘Hit-and-run programming’ because of the simplicity of the method. Interestingly, the optimization of the delivery was performed with eGFP mRNA.  This post describes how TriLink’s mRNAs contribute to the simple and efficient delivery of mRNA into cells.

In this article, the authors showed that mRNA delivery into cells can be simple and efficient thanks to their nanocarriers. Here is a new good example of the major role of mRNAs as New Biologic Entities (NBEs). And tebu-bio / TriLink mRNA production platform is convenient because:

mRNA-based gene editing to engineer anti-cancer T-cells

mRNA-based gene editing to engineer anti-cancer T-cells
mRNA-based gene editing to engineer anti-cancer T-cells

Classically, a megaTAL nuclease coding mRNA can be used to knock out genes in lymphocytes. Nevertheless, the CRISPR CAS9 system can also be used efficiently to disrupt the lymphocytes’ expression of T cell receptors. For that purpose, a new optimized CAS9 coding mRNA has been designed for this purpose. It has been modified to be more stable, be well accepted by the host cells and to translate more efficient CAS9 endonuclease. I invite you to read my post focused on that topic.

Adding a function can also be a way to program T cells and boost immunity against cancer cells. For that, the authors used a constitutively active FOXO1 protein coding mRNA. Indeed FOXO1 is key transcription factor of memory formation. It is involved in the transition in CD8 T-cells. They showed that the delivery of that mRNA can improve anti-tumor activities of the targeted T-cells. Thus, it avoids laborious in vitro stimulation-expansion.

Moffett et al, using their nanocarriers, concluded that mRNA delivery ensures efficient gene editing and programming of Chimeric Antigen Receptor T-cells (CAR-T-cells). This provides interesting leads for the future development of Adoptive T Cell Therapies (ACT), notably for T cell harvest from peripheral blood lymphocytes that require antigen-specific expansion or genetic engineering.

Hematopoietic stem cell research benefits from mRNA improvements

Cell therapy is now standard of care for thalassemia. Gamma-globulin gene insertion into hematopoietic stem cell (HSC) transplant is the key to reverse thalassemia. Conventionally, the gene insertion is done by virus vectors.

Here again, a vector-free delivery of the CRISPR CAS9 could reverse a mutation by knock-in. The addition of a ssODN (single stranded oligonucleotide) is required to trigger the Homology Repair mechanism at the center of the knock-in process.

Nevertheless, there is another issue with hematopoietic stem cells (HSC). They are very limited and the required propagation in vitro generally fails. Thus, Moffett et al tested Musashi-2 coding mRNA into HSC. Indeed, the protein is a key regulator of stem cells. They have shown that the transient expression through the mRNA is sufficient to boost the self-renewal capacity of the HSC.

It illustrates that the possibilities to use mRNA in cell therapy are very large. Furthermore mRNA should become a major tool for stem cell research in the next few months.

3 top improvements of mRNA for in vivo application into Mammalians cells

In their article, Moffett et al used 3 mRNA modifications: ARCA, 5meC and PseudoU. They are the conventional ones emerging since about 2010.

ARCA ensures the mRNA capping. Unfortunately it leads to a CAP0 structure. Today, thanks to the Trilink Biotechnologies’ developments, mRNA can be capped easily with a natural capping structure CAP1. The corresponding CleanCap technology also provides a capping efficiency around 97%. Furthermore the CAP1 structure boosts the translation and so delivers more functional protein into the cells.

5meC and PseudoU are well-known as modifications leading to reduction of the immune response of the cells against the exogenus mRNA. These modifications are covered by patents. So, Trilink Biotechnologies bring a license-free alternative that ultimately is even more efficient in reducing the immune response of the cells. It’s the 5moU modification and the reduction of cytotoxicity as illustrated just below.

5moU mRNA reduce cytotoxicity
Reduce cytotoxicity with 5moU moidfied mRNA

Below there is an illustration of the eGFP fluorescence measured in several cell types transfected with wild type mRNA or modified mRNA.

GFP fluorescence boosted by modified mRNA
GFP fluorescence boosted with 5moU modification into several cell types

The 3 top modifications for mRNA:

  • CleanCap providing high capping efficiency and CAP1 structure
  • 5-Methoxy-UTP
  • U-depleted sequence, as per our CAS9 mRNA

Taking all the above points together, these improvements make mRNA a simple and reliable tool, as a promising alternative to virus vectors for therapies.

For further information on TriLink’s mRNA (or your CRISPR CAS9 gene editing projects in general), please don’t hesitate to contact me by leaving your comments below, I’ll be pleased to answer.




2 Responses

    1. The mRNA is spliced.
      Actually, the coding sequence (ORF) is directly cloned into an expression vector that includes optimized UTRs (in both 5′ and 3′). Thus, the mRNA resulting of the in vitro transcription is similar to a mature/spliced mRNA. The structure can be illustrated as Cap1-UTR-ORF-UTR(with 120A).

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