Local Delivery ofOx40l,Cd80, andCd86mRNA Kindles Global Anticancer Immunity

Haabeth, Ole Audun Werner; Blake, Timothy R.; McKinlay, Colin J.; Tveita, Anders Aune; Sallets, Adrienne; Waymouth, Robert M.; Wender, Paul A.; Levy, Ronald

doi:10.1158/0008-5472.can-18-2867

Cited by 100 publications

(99 citation statements)

References 47 publications

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“…1) that are effective for the delivery of mRNA and pDNA to a variety of cell types in cell culture and live animals. 33,[45][46][47][48][49] These cationic amphiphilic oligomers, when complexed with negatively-charged oligonucleotides, form polyelectrolyte complexes that exhibit dynamic, charge-altering behavior that facilitates the intracellular release of the oligonucleotides. 33 This dynamic charge-altering behavior was attributed to the unique structural features of the cationic a-aminoester sequences 44 of these amphiphilic oligomers which exhibit a pH-dependent selective degradation to neutral diketopiperazines.…”

Section: Discussionmentioning

confidence: 99%

“…44 We have leveraged this behavior in the design of novel cationic amphiphilic oligomers, charge-altering releasable transporters (CARTs), 33,[45][46][47] that bind, complex, and deliver oligonucleotides of wide ranging size (mRNA, pDNA) into cells, in vitro and in vivo. 33,[45][46][47][48][49] At low pH, these oligomeric amphiphiles are cationic and associate in water with polyanionic oligonucleotides to form polyelectrolyte complexes. At elevated pH these polyelectrolyte complexes release the oligonucleotides, which was attributed to the charge-altering degradative behavior of the cationic oligo(aamino ester)s (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…These charge-altering releasable transporters (CARTs) 33,[45][46][47] were shown to be effective for mRNA and pDNA delivery to a variety of cell types in cell culture and live animals, enabling multiple therapeutic vaccination strategies for eradication of established tumors in mice. 48,49 As the pH-induced charge-canceling degradation of the cationic poly(a-aminoester) backbone was implicated in the intracellular release of mRNA, 33 herein we describe a detailed mechanistic study directed at elucidation of the structural and environmental factors that lead to the selective degradation processes for the cationic poly(ammonium ester) homopolymers in aqueous buffers. While not directly comparable to the polyelectrolyte environments in which the amphiphilic CARTs degrade, 33,[45][46][47] these studies provide fundamental insights on the structural factors that contribute to the degradation behavior of poly(amino ester)s in aqueous buffers.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis and mechanistic investigations of pH-responsive cationic poly(aminoester)s

Blake

Turlington

et al. 2020

Chem. Sci.

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The synthesis and degradation mechanisms of a class of pH-sensitive, rapidly degrading cationic poly(aaminoester)s are described. These reactive, cationic polymers are stable at low pH in water, but undergo a fast and selective degradation at higher pH to liberate neutral diketopiperazines. Related materials incorporating oligo(a-amino ester)s have been shown to be effective gene delivery agents, as the charge-altering degradative behavior facilitates the delivery and release of mRNA and other nucleic acids in vitro and in vivo. Herein, we report detailed studies of the structural and environmental factors that lead to these rapid and selective degradation processes in aqueous buffers. At neutral pH, poly(aaminoester)s derived from N-hydroxyethylglycine degrade selectively by a mechanism involving sequential 1,5-and 1,6-O/N acyl shifts to generate bis(N-hydroxyethyl) diketopiperazine. A family of structurally related cationic poly(aminoester)s was generated to study the structural influences on the degradation mechanism, product distribution, and pH dependence of the rate of degradation. The kinetics and mechanism of the pH-induced degradations were investigated by 1 H NMR, model reactions, and kinetic simulations. These results indicate that polyesters bearing a-ammonium groups and appropriately positioned N-hydroxyethyl substituents are readily cleaved (by intramolecular attack) or hydrolyzed, representing dynamic "dual function" materials that are initially polycationic and transform with changing environment to neutral products. Fig. 1 Proposed mRNA binding and release by Charge-Altering Releasable Transporters (CARTs). Selective degradation of the polycationic poly(a-aminoester) block generates neutral diketopiperazines and hydrolyzed N-hydroxyethyl glycines.Fig. 2 Synthesis of azalactone monomers and polymers.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Synthesis and mechanistic investigations of pH-responsive cationic poly(aminoester)s

Blake

Turlington

et al. 2020

Chem. Sci.

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“…However, through high-throughput screening, we previously discovered that CARTs containing both unsaturated C18 oleyl and C9 nonenyl lipids (CART O 5 :N 6 :A 9 ( 2 ), CART BDK-O 7 :N 7 :A 13 ( 3 )) resulted in enhanced transfection of T and B lymphocytes, significantly outperforming single-lipid CARTs and commercial reagents 41 . In fact, these mixed-lipid CARTs resulted in >80% transfection of immortalized T lymphocytes in vitro 41 as well as ≥1.5% in vivo in living mice when administered intravenously 46,52 , significantly outperforming previous transfection technologies for mRNA delivery into T lymphocytes 41,53–55 . We therefore hypothesized that CARTs may represent a novel delivery vector for highly transfection-recalcitrant resting NK cells.…”

Section: Resultsmentioning

confidence: 96%

“…Further studies will need to examine whether sufficient numbers of CART-generated CAR NK cells can be readily generated, and to assess their lifespan in vivo in order to determine if this technique would be superior to lentiviral transduction for CAR NK cell preparation. As we have previously demonstrated that CARTs can be used to deliver mRNA in vivo with a high specificity for mRNA expression in the spleen 41,46,52 , in vivo CART-mediated delivery of CAR-encoding mRNA may provide an alternative therapeutic approach for the generation of CAR NK cells as a strategy for personalized cancer immunotherapy.…”

Section: Discussionmentioning

confidence: 99%

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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