skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

We report a strategy for generating a combinatorial library of oligonucleotide transporters with varied lipid domains and their use in the efficient transfection of lymphocytes with mRNA in vitro and in vivo. This library is based on amphiphilic charge-altering releasable transporters (CARTs) that contain a lipophilic block functionalized with various side-chain lipids and a polycationic α-amino ester mRNA-binding block that undergoes rearrangement to neutral small molecules, resulting in mRNA release. We show that certain binary mixtures of these lipid-varied CARTs provide up to a ninefold enhancement in mRNA translation in lymphocytes in vitro relative to either a single-lipid CART component alone or the commercial reagent Lipofectamine 2000, corresponding to a striking increase in percent transfection from 9–12% to 80%. Informed by the results with binary mixtures, we further show that CARTs consisting of optimized ratios of the two lead lipids incorporated into a single hybrid-lipid transporter molecule maintain the same delivery efficacy as the noncovalent mixture of two CARTs. The lead lipid CART mixtures and hybrid-lipid CARTs show enhanced lymphocyte transfection in primary T cells and in vivo in mice. This combinatorial approach for rapidly screening mRNA delivery vectors has provided lipid-varied CART mixtures and hybrid-lipid CARTs that exhibit significantmore » improvement in mRNA delivery to lymphocytes, a finding of potentially broad value in research and clinical applications.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division; National Institutes of Health (NIH)
OSTI Identifier:
1441127
Alternate Identifier(s):
OSTI ID: 1540298; OSTI ID: 1594176
Grant/Contract Number:  
SC0018168; CHE-1607092; NIH-CA031841; NIH-CA031845; S10RR027431-01
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 26; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; gene therapy; nanoparticle; polyplex; combinatorial; immunotherapy

Citation Formats

McKinlay, Colin J., Benner, Nancy L., Haabeth, Ole A., Waymouth, Robert M., and Wender, Paul A. Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters. United States: N. p., 2018. Web. doi:10.1073/pnas.1805358115.
McKinlay, Colin J., Benner, Nancy L., Haabeth, Ole A., Waymouth, Robert M., & Wender, Paul A. Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters. United States. doi:10.1073/pnas.1805358115.
McKinlay, Colin J., Benner, Nancy L., Haabeth, Ole A., Waymouth, Robert M., and Wender, Paul A. Mon . "Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters". United States. doi:10.1073/pnas.1805358115.
@article{osti_1441127,
title = {Enhanced mRNA delivery into lymphocytes enabled by lipid-varied libraries of charge-altering releasable transporters},
author = {McKinlay, Colin J. and Benner, Nancy L. and Haabeth, Ole A. and Waymouth, Robert M. and Wender, Paul A.},
abstractNote = {We report a strategy for generating a combinatorial library of oligonucleotide transporters with varied lipid domains and their use in the efficient transfection of lymphocytes with mRNA in vitro and in vivo. This library is based on amphiphilic charge-altering releasable transporters (CARTs) that contain a lipophilic block functionalized with various side-chain lipids and a polycationic α-amino ester mRNA-binding block that undergoes rearrangement to neutral small molecules, resulting in mRNA release. We show that certain binary mixtures of these lipid-varied CARTs provide up to a ninefold enhancement in mRNA translation in lymphocytes in vitro relative to either a single-lipid CART component alone or the commercial reagent Lipofectamine 2000, corresponding to a striking increase in percent transfection from 9–12% to 80%. Informed by the results with binary mixtures, we further show that CARTs consisting of optimized ratios of the two lead lipids incorporated into a single hybrid-lipid transporter molecule maintain the same delivery efficacy as the noncovalent mixture of two CARTs. The lead lipid CART mixtures and hybrid-lipid CARTs show enhanced lymphocyte transfection in primary T cells and in vivo in mice. This combinatorial approach for rapidly screening mRNA delivery vectors has provided lipid-varied CART mixtures and hybrid-lipid CARTs that exhibit significant improvement in mRNA delivery to lymphocytes, a finding of potentially broad value in research and clinical applications.},
doi = {10.1073/pnas.1805358115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 26,
volume = 115,
place = {United States},
year = {2018},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1073/pnas.1805358115

Citation Metrics:
Cited by: 11 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Gene therapy clinical trials worldwide to 2012 - an update: Gene therapy clinical trials worldwide to 2012 - an update
journal, February 2013

  • Ginn, Samantha L.; Alexander, Ian E.; Edelstein, Michael L.
  • The Journal of Gene Medicine, Vol. 15, Issue 2
  • DOI: 10.1002/jgm.2698

Towards personalized, tumour-specific, therapeutic vaccines for cancer
journal, December 2017

  • Hu, Zhuting; Ott, Patrick A.; Wu, Catherine J.
  • Nature Reviews Immunology, Vol. 18, Issue 3
  • DOI: 10.1038/nri.2017.131

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes
journal, March 2013


T Cell-Specific siRNA Delivery Using Antibody-Conjugated Chitosan Nanoparticles
journal, May 2012

  • Lee, Jangwook; Yun, Kyoung-Soo; Choi, Chang Seon
  • Bioconjugate Chemistry, Vol. 23, Issue 6
  • DOI: 10.1021/bc2006219

Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA
journal, December 2016

  • Miller, Jason B.; Zhang, Shuyuan; Kos, Petra
  • Angewandte Chemie International Edition, Vol. 56, Issue 4
  • DOI: 10.1002/anie.201610209

Global Manufacturing of CAR T Cell Therapy
journal, March 2017

  • Levine, Bruce L.; Miskin, James; Wonnacott, Keith
  • Molecular Therapy - Methods & Clinical Development, Vol. 4
  • DOI: 10.1016/j.omtm.2016.12.006

Development of Guanidinium-Rich Protein Mimics for Efficient siRNA Delivery into Human T Cells
journal, September 2015


Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy
journal, June 2016

  • Kranz, Lena M.; Diken, Mustafa; Haas, Heinrich
  • Nature, Vol. 534, Issue 7607
  • DOI: 10.1038/nature18300

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy
journal, December 2016


In Vitro and in Vivo mRNA Delivery Using Lipid-Enveloped pH-Responsive Polymer Nanoparticles
journal, April 2011

  • Su, Xingfang; Fricke, Jennifer; Kavanagh, Daniel G.
  • Molecular Pharmaceutics, Vol. 8, Issue 3
  • DOI: 10.1021/mp100390w

Clinical manufacturing of CAR T cells: foundation of a promising therapy
journal, January 2016

  • Wang, Xiuyan; Rivière, Isabelle
  • Molecular Therapy - Oncolytics, Vol. 3
  • DOI: 10.1038/mto.2016.15

mRNA-based therapeutics — developing a new class of drugs
journal, September 2014

  • Sahin, Ugur; Karikó, Katalin; Türeci, Özlem
  • Nature Reviews Drug Discovery, Vol. 13, Issue 10
  • DOI: 10.1038/nrd4278

Systemic mRNA Delivery to the Lungs by Functional Polyester-based Carriers
journal, November 2017


Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity☆
journal, December 2006


In Situ Functionalized Polymers for siRNA Delivery
journal, April 2016

  • Priegue, Juan M.; Crisan, Daniel N.; Martínez-Costas, José
  • Angewandte Chemie International Edition, Vol. 55, Issue 26
  • DOI: 10.1002/anie.201601441

Lipid composition of cationic nanoliposomes implicate on transfection efficiency
journal, March 2013


Toxicity of cationic lipids and cationic polymers in gene delivery
journal, August 2006


A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery
journal, June 2008

  • Green, Jordan J.; Langer, Robert; Anderson, Daniel G.
  • Accounts of Chemical Research, Vol. 41, Issue 6
  • DOI: 10.1021/ar7002336

Transfecting the hard-to-transfect lymphoma/leukemia cells using a simple cationic polymer nanocomplex
journal, April 2012


Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells
journal, May 1999


Materials for non-viral intracellular delivery of messenger RNA therapeutics
journal, October 2016


Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes
journal, November 2002

  • McManus, Michael T.; Haines, Brian B.; Dillon, Christopher P.
  • The Journal of Immunology, Vol. 169, Issue 10
  • DOI: 10.4049/jimmunol.169.10.5754

Thiourea-Based Bifunctional Organocatalysis:  Supramolecular Recognition for Living Polymerization
journal, October 2005

  • Dove, Andrew P.; Pratt, Russell C.; Lohmeijer, Bas G. G.
  • Journal of the American Chemical Society, Vol. 127, Issue 40
  • DOI: 10.1021/ja0543346

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery
journal, February 2016

  • Fenton, Owen S.; Kauffman, Kevin J.; McClellan, Rebecca L.
  • Advanced Materials, Vol. 28, Issue 15
  • DOI: 10.1002/adma.201505822

An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo
journal, November 2015


Nanoparticulate Nonviral Agent for the Effective Delivery of pDNA and siRNA to Differentiated Cells and Primary Human T Lymphocytes
journal, September 2012

  • Schallon, Anja; Synatschke, Christopher V.; Jérôme, Valérie
  • Biomacromolecules, Vol. 13, Issue 11
  • DOI: 10.1021/bm3012055

Functional DNA Delivery Enabled by Lipid-Modified Charge-Altering Releasable Transporters (CARTs)
journal, May 2018


Lipid-like materials for low-dose, in vivo gene silencing
journal, January 2010

  • Love, K. T.; Mahon, K. P.; Levins, C. G.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 5
  • DOI: 10.1073/pnas.0910603106

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition
journal, January 2017

  • Lostalé-Seijo, Irene; Louzao, Iria; Juanes, Marisa
  • Chemical Science, Vol. 8, Issue 12
  • DOI: 10.1039/C7SC03918B

Multi-Emissive Difluoroboron Dibenzoylmethane Polylactide Exhibiting Intense Fluorescence and Oxygen-Sensitive Room-Temperature Phosphorescence
journal, July 2007

  • Zhang, Guoqing; Chen, Jianbin; Payne, Sarah J.
  • Journal of the American Chemical Society, Vol. 129, Issue 29
  • DOI: 10.1021/ja0720255

Engineered T cells: the promise and challenges of cancer immunotherapy
journal, August 2016

  • Fesnak, Andrew D.; June, Carl H.; Levine, Bruce L.
  • Nature Reviews Cancer, Vol. 16, Issue 9
  • DOI: 10.1038/nrc.2016.97

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals
journal, January 2017

  • McKinlay, Colin J.; Vargas, Jessica R.; Blake, Timothy R.
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 4
  • DOI: 10.1073/pnas.1614193114

Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization
journal, September 2016


Modulated Protonation of Side Chain Aminoethylene Repeats in N-Substituted Polyaspartamides Promotes mRNA Transfection
journal, August 2014

  • Uchida, Hirokuni; Itaka, Keiji; Nomoto, Takahiro
  • Journal of the American Chemical Society, Vol. 136, Issue 35
  • DOI: 10.1021/ja506194z

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA
journal, August 2017


mRNA vaccines — a new era in vaccinology
journal, January 2018

  • Pardi, Norbert; Hogan, Michael J.; Porter, Frederick W.
  • Nature Reviews Drug Discovery, Vol. 17, Issue 4
  • DOI: 10.1038/nrd.2017.243

Cationic liposome-mediated RNA transfection.
journal, August 1989

  • Malone, R. W.; Felgner, P. L.; Verma, I. M.
  • Proceedings of the National Academy of Sciences, Vol. 86, Issue 16
  • DOI: 10.1073/pnas.86.16.6077

Transfection efficiency boost of cholesterol-containing lipoplexes
journal, September 2012

  • Pozzi, Daniela; Marchini, Cristina; Cardarelli, Francesco
  • Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1818, Issue 9
  • DOI: 10.1016/j.bbamem.2012.05.017

Bulk antimony sulfide with excellent cycle stability as next-generation anode for lithium-ion batteries
journal, April 2014

  • Yu, Denis Y. W.; Hoster, Harry E.; Batabyal, Sudip K.
  • Scientific Reports, Vol. 4, Issue 1
  • DOI: 10.1038/srep04562

Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems
journal, January 2017


Rational design of cationic lipids for siRNA delivery
journal, January 2010

  • Semple, Sean C.; Akinc, Akin; Chen, Jianxin
  • Nature Biotechnology, Vol. 28, Issue 2
  • DOI: 10.1038/nbt.1602

Lymphocyte apoptosis: induction by gene transfer techniques
journal, April 1997


Polyamine-Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery
journal, September 2017

  • Li, Jiahe; He, Yanpu; Wang, Wade
  • Angewandte Chemie International Edition, Vol. 56, Issue 44
  • DOI: 10.1002/anie.201707466

Dynamic Amphiphile Libraries To Screen for the “Fragrant” Delivery of siRNA into HeLa Cells and Human Primary Fibroblasts
journal, June 2013

  • Gehin, Charlotte; Montenegro, Javier; Bang, Eun-Kyoung
  • Journal of the American Chemical Society, Vol. 135, Issue 25
  • DOI: 10.1021/ja404153m

In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers
journal, April 2017

  • Smith, Tyrel T.; Stephan, Sirkka B.; Moffett, Howell F.
  • Nature Nanotechnology, Vol. 12, Issue 8
  • DOI: 10.1038/nnano.2017.57

Tagging alcohols with cyclic carbonate: a versatile equivalent of (meth)acrylate for ring-opening polymerization
journal, January 2008

  • Pratt, Russell C.; Nederberg, Fredrik; Waymouth, Robert M.
  • Chem. Commun., Issue 1
  • DOI: 10.1039/B713925J

Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity
journal, May 2010


Effects of local structural transformation of lipid-like compounds on delivery of messenger RNA
journal, February 2016

  • Li, Bin; Luo, Xiao; Deng, Binbin
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep22137

Gene therapy returns to centre stage
journal, October 2015


Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes
journal, July 2017

  • Fenton, Owen S.; Kauffman, Kevin J.; Kaczmarek, James C.
  • Advanced Materials, Vol. 29, Issue 33
  • DOI: 10.1002/adma.201606944

Organocatalytic Ring-Opening Polymerization of Morpholinones: New Strategies to Functionalized Polyesters
journal, June 2014

  • Blake, Timothy R.; Waymouth, Robert M.
  • Journal of the American Chemical Society, Vol. 136, Issue 26
  • DOI: 10.1021/ja503830c

A brief history of gene therapy
journal, October 1992


Novel Protein Transduction Domain Mimics as Nonviral Delivery Vectors for siRNA Targeting NOTCH1 in Primary Human T cells
journal, January 2013

  • Tezgel, A. Özgül; Gonzalez-Perez, Gabriela; Telfer, Janice C.
  • Molecular Therapy, Vol. 21, Issue 1
  • DOI: 10.1038/mt.2012.209

Engineering liposomal nanoparticles for targeted gene therapy
journal, May 2017

  • Zylberberg, C.; Gaskill, K.; Pasley, S.
  • Gene Therapy, Vol. 24, Issue 8
  • DOI: 10.1038/gt.2017.41

Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells
journal, July 2012

  • Geihe, E. I.; Cooley, C. B.; Simon, J. R.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 33
  • DOI: 10.1073/pnas.1211361109

A new developing class of gene delivery: messenger RNA-based therapeutics
journal, January 2017

  • Meng, Zhao; O'Keeffe-Ahern, Jonathan; Lyu, Jing
  • Biomaterials Science, Vol. 5, Issue 12
  • DOI: 10.1039/C7BM00712D

mRNA-based therapeutics–Advances and perspectives
journal, July 2016

  • Sergeeva, O. V.; Koteliansky, V. E.; Zatsepin, T. S.
  • Biochemistry (Moscow), Vol. 81, Issue 7
  • DOI: 10.1134/S0006297916070075

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics
journal, April 2008

  • Akinc, Akin; Zumbuehl, Andreas; Goldberg, Michael
  • Nature Biotechnology, Vol. 26, Issue 5
  • DOI: 10.1038/nbt1402

Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines
journal, August 2012


High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation
journal, January 2006


mRNA vaccine delivery using lipid nanoparticles
journal, May 2016

  • Reichmuth, Andreas M.; Oberli, Matthias A.; Jaklenec, Ana
  • Therapeutic Delivery, Vol. 7, Issue 5
  • DOI: 10.4155/tde-2016-0006

Polycation-based nanoparticle delivery of RNAi therapeutics: Adverse effects and solutions
journal, December 2012

  • Ballarín-González, Borja; Howard, Kenneth Alan
  • Advanced Drug Delivery Reviews, Vol. 64, Issue 15
  • DOI: 10.1016/j.addr.2012.07.004

Messenger RNA-based therapeutics for the treatment of apoptosis-associated diseases
journal, October 2015

  • Matsui, Akitsugu; Uchida, Satoshi; Ishii, Takehiko
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep15810

Structurally Programmed Assembly of Translation Initiation Nanoplex for Superior mRNA Delivery
journal, February 2017


Organocatalysis: Opportunities and Challenges for Polymer Synthesis
journal, March 2010

  • Kiesewetter, Matthew K.; Shin, Eun Ji; Hedrick, James L.
  • Macromolecules, Vol. 43, Issue 5
  • DOI: 10.1021/ma9025948