Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening

JOURNAL OF CONTROLLED RELEASE

卷:316

Article

Guimaraes, Pedro P. G.^1,2  Zhang, Rui¹  Spektor, Roman³  Tan, Mingchee¹  Chung, Amanda¹  Billingsley, Margaret M.¹  El-Mayta, Rakan¹  Riley, Rachel S.¹  Wang, Lili⁴  Wilson, James M.⁴  Mitchell, Michael J.^1,5,6,7,8 

[1] Univ Penn, Dept Bioengn, 240 Skirkanich Hall,210 S 33rd St, Philadelphia, PA 19104 USA

[2] Univ Fed Minas Gerais, Inst Biol Sci, Dept Physiol & Biophys, Belo Horizonte, MG, Brazil

[3] Cornell Univ, Grad Field Genet Genom & Dev, Ithaca, NY USA

[4] Univ Penn, Dept Med, Perelman Sch Med, Gene Therapy Program, Philadelphia, PA 19104 USA

[5] Univ Penn, Perelman Sch Med, Abramson Canc Ctr, Philadelphia, PA 19104 USA

[6] Univ Penn, Perelman Sch Med, Inst Immunol, Philadelphia, PA 19104 USA

[7] Univ Penn, Perelman Sch Med, Cardiovasc Inst, Philadelphia, PA 19104 USA

[8] Univ Penn, Perelman Sch Med, Inst Regenerat Med, Philadelphia, PA 19104 USA

关键词: mRNA;    Nanoparticle;    Gene delivery;    Gene therapy;    High-throughput screening;   

DOI  :  10.1016/j.jconrel.2019.10.028

来源: Elsevier

【 摘 要 】

Messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapy, with the potential to induce protein production to treat and prevent a range of diseases. However, the widespread use of mRNA as a therapeutic requires safe and effective in vivo delivery technologies. Libraries of ionizable lipid nanoparticles (LNPs) have been designed to encapsulate mRNA, prevent its degradation, and mediate intracellular delivery. However, these LNPs are typically characterized and screened in an in vitro setting, which may not fully replicate the biological barriers that they encounter in vivo. Here, we designed and evaluated a library of engineered LNPs containing barcoded mRNA (b-mRNA) to accelerate the screening of mRNA delivery platforms in vivo. These b-mRNA are similar in structure and function to regular mRNA, and contain barcodes that enable their delivery to be quantified via deep sequencing. Using a mini-library of b-mRNA LNPs formulated via microfluidic mixing, we show that these different formulations can be pooled together, administered intravenously into mice as a single pool, and their delivery to multiple organs (liver, spleen, brain, lung, heart, kidney, pancreas, and muscle) can be quantified simultaneously using deep sequencing. In the context of liver and spleen delivery, LNPs that exhibited high b-mRNA delivery also yielded high luciferase expression, indicating that this platform can identify lead LNP candidates as well as optimal formulation parameters for in vivo mRNA delivery. Interestingly, LNPs with identical formulation parameters that encapsulated different types of nucleic acid barcodes (b-mRNA versus a DNA barcode) altered in vivo delivery, suggesting that the structure of the barcoded nucleic acid affects LNP in vivo delivery. This platform, which enables direct barcoding and subsequent quantification of a functional mRNA, can accelerate the in vivo screening and design of LNPs for mRNA therapeutic applications such as CRISPR-Cas9 gene editing, mRNA vaccination, and other mRNA-based regenerative medicine and protein replacement therapies.

【 授权许可】

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