Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

McKinlay, Colin J.; Benner, Nancy L.; Haabeth, Ole Audun Werner; Waymouth, Robert M.; Wender, Paul A.

doi:10.1073/pnas.1805358115

Cited by 187 publications

(216 citation statements)

References 69 publications

(105 reference statements)

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“…The first generation single-lipid CART D 13 :A 11 ( 1 ) has been used for efficient mRNA delivery in vitro (>95% transfection in many cultured cells) and in vivo ( Figure 1A ) 44 . However, decreased transfection efficiencies were observed in T lymphocytes compared to other cell types, a common trend among transfection reagents 41,50 . Lymphocytes are hypothesized to be difficult for transfection by viral and non-viral vectors in part due to their limited endocytosis and protein translation as well as the detrimental effect of transfection reagents on their viability 51 .…”

Section: Resultsmentioning

confidence: 98%

“…CARTs are synthetic, biodegradable and dynamic delivery vectors, based on di- or tri-block oligomers, consisting of a lipid block(s) followed by a charge-altering block ( Figure 1A ) 41–44 . The charge-altering block is initially cationic for mRNA complexation but rearranges to neutral diketopiperazine small molecules at pH 7.4 facilitating the release of the anionic mRNA while avoiding toxicities associated with persistent cations 41,48,49 . The first generation single-lipid CART D 13 :A 11 ( 1 ) has been used for efficient mRNA delivery in vitro (>95% transfection in many cultured cells) and in vivo ( Figure 1A ) 44 .…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Recently, we reported the use of Charge-Altering Releasable Transporters (CARTs) for mRNA delivery into T and B lymphocytes 41 . CARTs are multi-block oligomers consisting of a lipid block(s) and a charge-altering block 41–47 . CARTs are initially cationic serving to complex polyanionic nucleic acids but rearrange under basic conditions (pH 7.4) to neutral products, facilitating the release of the anionic cargo 44,47 .…”

Section: Introductionmentioning

confidence: 99%

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Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Wilk

Benner

Vergara

et al. 2020

Preprint

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22 Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools 23 for immunotherapy. However, their recalcitrance to standard transfection techniques has limited 24 both mechanistic studies and clinical applications. Current approaches for NK cell manipulation 25 rely on viral transduction or methods requiring NK cell activation, which can alter NK cell 26 function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) 27 efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. 28 Compared to electroporation, CARTs transfect NK cells two orders of magnitude more 29 efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype 30 and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric 31 antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our 32 knowledge, CARTs represent the first efficacious transfection technique for resting primary NK 33 cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical 34 applications of this understudied lymphocyte subset. 35 36 37 38 Natural killer (NK) cells are a group of innate lymphocytes that coordinate and execute 39 the rapid elimination of neoplastic and virus-infected cells 1,2 . Upon activation through a 40 combinatorial array of germline-encoded inhibitory and activating receptors, NK cells can 41 directly kill their targets via targeted release of perforin-and granzyme-containing granules, as 42 well as coordinate the downstream immune response by secreting pro-inflammatory cytokines 43 like IFNγ and TNFα 3,4 . The rapid and robust cytotoxicity of NK cells makes them excellent 44 assets for antiviral and anticancer immunotherapy, an enthusiasm most recently bolstered by 45 the use of chimeric antigen receptor (CAR) NK cells in treating CD19 + lymphoid cancers with 46 high efficacy and low toxicity 5-13 . Despite their clinical promise, several fundamental facets of 47 human NK cell biology are poorly understood. For example, the molecular underpinnings of 48 human NK cell memory 14-16 and the precise roles of various NK cell receptors in target cell 49 recognition remain unknown. 50 NK cells are notoriously difficult to manipulate, presenting challenges both for a deeper 51 understanding of fundamental NK cell biology and for the use of NK cells in clinical 52 applications 17-21 . Most clinical trials involving genetically-modified NK cells utilize viral 53 transduction (NCT02944162, NCT02892695, NCT03056339, NCT03579927) 5,22 . While viral 54 transduction enables stable transgene expression, it is costly, laborious, and induces robust NK 55 cell activation and apoptosis due to triggering of innate nucleic acid sensors, limiting its 56 practicality for mechanistic studies of NK cell biology 8,18,23-28 . Further, in clinical applications, 57 viral transduction carries the risks of sustained adverse effects and oncogenic potential ...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Wilk

Benner

Vergara

et al. 2020

Preprint

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“…However,m ajor challenges exist in efficient delivery of mRNAtohard-to-transfect cells (for example,macrophages) and the underlying reason is not clear. [2b, 4] Current mRNA delivery systems primarily focus on cellular delivery functions, [3,5] through tuning nanoparticle size [6] to enhance cellular uptake,a djusting surface charge to control the loading/release profile of mRNA [7] and biodistributions. [8] However,f ew provide the functionality to directly regulate the mRNAt ranslation process.…”

mentioning

confidence: 99%

Functional Nanoparticles with a Reducible Tetrasulfide Motif to Upregulate mRNA Translation and Enhance Transfection in Hard‐to‐Transfect Cells

et al. 2020

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Effective messenger RNA( mRNA) transfection in hard-to-transfect cells delivered by vectors is al ong-standing challenge.N ow it is hypothesizedt hat the high intracellular glutathione level is associated with suppressed mRNAt ranslation. This theory leads to an ew design principle of nextgeneration mRNAv ectors:n anoparticles with glutathione depletion chemistry upregulate mRNAt ranslation and enhance transfection, which is beneficial for mRNAd elivery in hard-to-transfect cells in vitro and in vivo.

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Self‐Degradable Lipid‐Like Materials Based on “Hydrolysis accelerated by the intra‐Particle Enrichment of Reactant (HyPER)” for Messenger RNA Delivery

Tanaka

Takahashi

Konishi

et al. 2020

Adv Funct Materials

106

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RNA-based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self-degradable lipid-like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self-degradability is based on a unique reaction "Hydrolysis accelerated by intra-Particle Enrichment of Reactant (HyPER)." In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid-scaffold lipid-like material that mounts all of these units (ssPalmO-Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.

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Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters

Cited by 187 publications

References 69 publications

Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Functional Nanoparticles with a Reducible Tetrasulfide Motif to Upregulate mRNA Translation and Enhance Transfection in Hard‐to‐Transfect Cells

Self‐Degradable Lipid‐Like Materials Based on “Hydrolysis accelerated by the intra‐Particle Enrichment of Reactant (HyPER)” for Messenger RNA Delivery

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