Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Liposomes are effective for delivering messenger RNA (mRNA) into cells.
• Co-delivering multiple mRNA types into individual cells presents a significant challenge for precise protein expression control.

Purpose of the Study:

• To investigate methods for efficient co-delivery of multiple mRNA types into individual cells.
• To compare the efficacy of mingle-lipoplexes versus single-lipoplexes for ratiometric mRNA delivery.
• To develop a theoretical framework explaining mRNA delivery stochasticity.

Main Methods:

• Co-delivery of varying ratiometric doses of mRNA encoding red (R) and green (G) fluorescent proteins using liposomes.
• Comparison of mingle-lipoplexes (multiple mRNAs in one lipoplex) and single-lipoplexes (one mRNA per lipoplex).
• Quantitative analysis using a theoretical framework modeling mRNA uptake and endosomal escape.
• Validation in 3D retinal organoids and zebrafish embryos.

Main Results:

• Packaging multiple mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial for efficient, ratio-controlled delivery into individual cells.
• Single-lipoplexes result in significant cell-to-cell variation in mRNA uptake and expression, achieving ratiometric dosing only at the population level.
• A theoretical framework quantitatively explains the observed differences in delivery efficiency and stochasticity.
• Mingle-lipoplexes demonstrate superior performance in delivering both mRNA types into single cells in complex biological models.

Conclusions:

• Mingle-lipoplexes enable precise control over co-delivered mRNA ratios at the single-cell level.
• This approach overcomes the limitations of single-lipoplexes, which exhibit high variability in multi-mRNA delivery.
• The findings are critical for applications requiring strict control of protein expression in individual cells, such as gene therapy and synthetic biology.