In the era of cell and gene therapy, the exponential increase of articles covering delivery methods over the past few years demonstrates the critical need for a cutting-edge technology to transfer, very efficiently but safely, CRISPR-Cas9 along with various coding mRNAs in the same particle.
What makes a delivery technology efficient ?
An efficient delivery system implies:
- A high penetration rate into target cells
- A high level of expression of the gene(s) of interest
- An all-in-one delivery system
- The preservation of the viability and the original cell phenotype
With these needs in mind, we have developed a new RNA delivery technology referred as LentiFlash®. Through a fast and transitory RNA expression, this non-integrative particle is particularly suitable for carrying coding and non-coding RNAs in all types of cells, while preserving the original cell phenotype and viability. We have also demonstrated that LentiFlash® particles are capable of transporting 5 to 6 RNA molecules per particle. These molecules can be identical or different, unlike an integrative lentiviral particle which is only capable of transporting 2 identical RNA molecules (Prel et al. Mol.Ther. 2015).
The data below, gathered by Flash Therapeutics, demonstrates that LentiFlash® is an all-in-one technology capable of delivering multiple sequences of interest in one simple step and using one single vector.
LentiFlash® efficiently carries several different types of RNA such as the CRISPR-Cas9 system in one particle.
HCT116-GFP cells are a stable cell clone (D2) expressing only one copy of the GFP gene that has been introduced with an integrative lentiviral vector expressing the GFP reporter. This model is used to evaluate improvements of the LentiFlash® particle carrying the CRISPR-Cas9 system.
HCT116-GFP clone is transduced with LentiFlash® carrying Cas9 and a sgRNA targeting the GFP gene. The GFP expression level is analyzed by flow cytometry.
Figure 1: Efficient GFP Knock-Out in clone D2 HCT116 cells using two subsequent LentiFlash® generations (data gathered by Flash Therapeutics).
This bar chart shows that the first LentiFlash® generation (v1) reaches 95% of KO with a dose of 5 pg p24/cell. When using the new LentiFlash® generation (v2) the same KO rate (>85%) is achieved using 5 times less particles (1 pg p24/cell).
LentiFlash® delivers safely 2 types of RNA molecules such as the CRISPR-Cas9 system, in one partcle to human primary T cells or in vivo (more data available on demand).
2- Vaccination : transduction of dendritic cells with different tumoral antigens and immunomodulators
LentiFlash® delivers several RNA encoding tumoral antigens or immunomodulators in human primary dendritic cells.
Human primary dendritic cells transduced with a LentiFlash® carrying 3 different human tumor associated antigens (TAAs) mRNAs: MAGE A3, gp100 and Tyrosinase. At 1 day post-transduction, the RNA content of the cells was quantified by RT-qPCR and compared to untransduced cells (NT).
Figure 2: Efficient delivery of 3 different tumoral antigens mRNAs (MAGEA3, GP100 and TYR) in human primary dendritic cells transduced by a LentiFlash® particle (data gathered by Flash Therapeutics).
This histogram shows that LentiFlash®-TAA allows efficient delivery of 3 different antigens into primary human Dendritic Cells.
Human primary dendritic cells have been transduced with a LentiFlash® carrying a human tumor associated antigen (TAA) mRNA MAGEA3, and the human interleukin-12 mRNA (IL-12). At 1 day post-transduction, the RNA content of the cells was quantified by RT-qPCR and compared to untransduced cells (NT).
Figure 3: Efficient delivery of an antigen (MAGEA3) and IL-12 mRNAs in human primary dendritic cells transduced by a LentiFlash® particle (data gathered by Flash Therapeutics).
This histogram shows that LentiFlash®-TAA/IL-12 allows efficient delivery in primary human Dendritic Cells of one mRNA for an antigen and another mRNA for an immunomodulatory molecule.
3- Stem cell differentiation
LentiFlash® delivers different RNAs such as transcription factor mRNAs to promote a cell-fate shift in human MSCs.
Highly immature primary human Mesenchymal Stem Cell-like cells (MSC-l) not expressing RUNX2 were transduced with LentiFlash® carrying RUNX2 mRNA or DLX5 mRNA (5 pg p24/cell). Controls cells were untransduced and BMP4-treated to differentiate MSC-l into functional osteoblasts. At 58 hours post-transduction, endogenous genomic RUNX2, DLX5, iBSP and OSTERIX mRNA levels were quantified by Real-Time quantitative PCR.
Figure 4: Efficient delivery of 2 different mRNAs in MSC-l cells transduced by LentiFlash®. Significance was measured by one-sample t-test with the reference value fixed to 1, which corresponds to MSC-l cells transduced by a control vector. Data are mean ± SD (n=4) (*P<0.05) (data published by Flash Therapeutics, Prel et al. Mol.Ther. 2015).
This histogram shows that when MSC-l cells are transduced by LentiFlash®-RUNX2 or DLX5, the endogenous RUNX2 and DLX5 mRNA levels significantly increased. In addition, the osteoblastic marker integrin-bone sialo-protein (iBSP) is upregulated in both conditions. Conversely, the osteoprogenitor master factor OSTERIX showed no change in expression after LentiFlash® transduction, suggesting a commitment of MSC-l towards an early osteoblastic lineage stage which is not surprising as the mRNA quantification was performed very early after the transduction (58H).