Biochemistry, Genetics and Molecular Biology › Molecular Biology

RNA Interference and Gene Delivery

Works Count: 94741

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This cluster of papers explores the mechanisms, delivery methods, and therapeutic applications of RNA interference (RNAi) in mammalian cells. It covers topics such as siRNA delivery, gene silencing, non-viral vectors, cell-penetrating peptides, lipid nanoparticles, and the potential of RNAi for gene therapy. The research also delves into the challenges and future directions of RNAi-based therapeutics.

Keywords

RNA Interference; siRNA Delivery; Gene Silencing; Non-viral Vectors; Therapeutic RNAi; Cell-penetrating Peptides; Lipid Nanoparticles; siRNA Therapeutics; Gene Therapy; Nucleic Acid Delivery

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells

2000-04-01

M. Lewin , Nadia Carlesso , Ching‐Hsuan Tung +4 more

Conditional gene targeting in macrophages and granulocytes using LysMcre mice.

1999-01-01

Björn E. Clausen , Christoph Burkhardt , Walter Reith +2 more

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

2001-05-01

Sayda M. Elbashir , Jens Harborth , Winfried Lendeckel +3 more

mRNA-based therapeutics — developing a new class of drugs

2014-09-19

Uğur Şahin , Katalin Karikó , Özlem Türeci

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Knocking down barriers: advances in siRNA delivery

2009-01-30

Kathryn A. Whitehead , Róbert Langer , Daniel G. Anderson

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Dicer is essential for mouse development

2003-10-05

Emily Bernstein , Sang Yong Kim , Michelle A. Carmell +7 more

‘Actively Acquired Tolerance’ of Foreign Cells

1953-10-01

R. E. Billingham , L Brent , P. B. Medawar

Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture

1977-11-01

Steven Collins , Robert C. Gallo , Robert E. Gallagher

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

1987-11-01

Philip L. Felgner , Thomas R. Gadek , Mai Marie Holm +6 more

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes … A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

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A review of stimuli-responsive nanocarriers for drug and gene delivery

2008-01-14

Srinivas Ganta , Harikrishna Devalapally , Aliasgar Shahiwala +1 more

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis

1991-07-01

Naoto Itoh , Shin Yonehara , Ai Ishii +6 more

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry

1991-06-01

Ildo Nicoletti , Graziella Migliorati , Maria Cristina Pagliacci +2 more

The nuclear RNase III Drosha initiates microRNA processing

2003-09-01

Yoontae Lee , Chiyoung Ahn , Jinju Han +7 more

Mechanisms of gene silencing by double-stranded RNA

2004-09-01

Gunter Meister , Thomas Tuschl

Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c- <i>MYC</i> transcription

2002-08-23

Adam Siddiqui-Jain , Cory L. Grand , David J. Bearss +1 more

The nuclease hypersensitivity element III 1 upstream of the P1 promoter of c- MYC controls 85–90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand … The nuclease hypersensitivity element III 1 upstream of the P1 promoter of c- MYC controls 85–90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c- MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c- MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c- MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c- MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

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Peptide therapeutics: current status and future directions

2014-10-17

Keld Fosgerau , Torsten Hoffmann

Peptides are recognized for being highly selective and efficacious and, at the same time, relatively safe and well tolerated. Consequently, there is an increased interest in peptides in pharmaceutical research … Peptides are recognized for being highly selective and efficacious and, at the same time, relatively safe and well tolerated. Consequently, there is an increased interest in peptides in pharmaceutical research and development (R&D), and approximately 140 peptide therapeutics are currently being evaluated in clinical trials. Given that the low-hanging fruits in the form of obvious peptide targets have already been picked, it has now become necessary to explore new routes beyond traditional peptide design. Examples of such approaches are multifunctional and cell penetrating peptides, as well as peptide drug conjugates. Here, we discuss the current status, strengths, and weaknesses of peptides as medicines and the emerging new opportunities in peptide drug design and development.

Nonviral Vectors for Gene Delivery

2008-12-03

Meredith A. Mintzer , Eric E. Simanek

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTNonviral Vectors for Gene DeliveryMeredith A. Mintzer and Eric E. Simanek*View Author Information Department of Chemistry, Texas A&M University, College Station, Texas 77843* To whom correspondence should … ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTNonviral Vectors for Gene DeliveryMeredith A. Mintzer and Eric E. Simanek*View Author Information Department of Chemistry, Texas A&M University, College Station, Texas 77843* To whom correspondence should be addressed. Tel.: (979) 845-4242. Fax: (979) 845-9452. E-mail: [email protected]Cite this: Chem. Rev. 2009, 109, 2, 259–302Publication Date (Web):December 3, 2008Publication History Received10 June 2008Published online3 December 2008Published inissue 11 February 2009https://pubs.acs.org/doi/10.1021/cr800409ehttps://doi.org/10.1021/cr800409ereview-articleACS PublicationsCopyright © 2008 American Chemical SocietyRequest reuse permissionsArticle Views25002Altmetric-Citations2038LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Chemical structure,Gene delivery,Genetics,Polyethylenimine,Toxicity Get e-Alerts

Toxicity of cationic lipids and cationic polymers in gene delivery

2006-05-14

Hongtao Lv , Shubiao Zhang , Bing Wang +2 more

Design and development of polymers for gene delivery

2005-07-01

Daniel W. Pack , Allan S. Hoffman , Suzie H. Pun +1 more

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

1995-08-01

Otmane Boussif , Frank Lezoualc’h , Maria Antonietta Zanta +4 more

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. … Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

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Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

2010-03-21

Mark E. Davis , Jonathan E. Zuckerman , Chung Hang Jonathan Choi +6 more

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Expression profiling reveals off-target gene regulation by RNAi

2003-05-16

Aimee L. Jackson , Steven R. Bartz , Janell M. Schelter +6 more

The dawning era of polymer therapeutics

2003-05-01

Ruth Duncan

A System for Stable Expression of Short Interfering RNAs in Mammalian Cells

2002-04-19

Thijn R. Brummelkamp , René Bernards , Reuven Agami

Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, … Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.

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Inducible Gene Targeting in Mice

1995-09-08

Ralf Kühn , Frieder Schwenk , Michel Aguet +1 more

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre … A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase β gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

Non-viral vectors for gene-based therapy

2014-07-15

Hao Yin , Rosemary Kanasty , Ahmed A. Eltoukhy +3 more

A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus

1997-06-01

Éric Vivès , Priscille Brodin , Bernard Lebleu

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through … Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an α-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the α-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nm. In contrast, a peptide with a full α-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 °C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest. Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an α-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the α-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nm. In contrast, a peptide with a full α-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 °C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest. Most "information-rich" molecules, such as oligonucleotides, genes, peptides, or proteins, are poorly taken up by cells since they do not efficiently cross the lipid bilayer of the plasma membrane or of the endocytic vesicles (Ref. 2Lebleu B. Trends Biotechnol. 1996; 14: 109-110Abstract Full Text PDF PubMed Scopus (29) Google Scholar, and references therein). This is considered to be a major limitation for their ex vivo orin vivo use in fundamental studies or in possible clinical applications. These compounds are currently delivered by various techniques including microinjection, electroporation, association with cationic lipids, liposome encapsidation, or receptor-mediated endocytosis. Various problems have been encountered in their use including low transfer efficiency, complex manipulation, cellular toxicity, or immunogenicity, which would preclude their routine usein vivo. As an alternative, several peptides have been successfully used to improve the intracellular delivery of nucleic acids or proteins. The fusogenic properties of influenza virus have been extensively studied in this context. They are currently assigned to a pH-dependent conformational change of the viral hemagglutinin leading to the exposure of its hydrophobic N-terminal region, and to the fusion of the viral and endosomal membranes (3Plank C. Oberhauser B. Mechtler K. Koch C. Wagner E. J. Biol. Chem. 1994; 269: 12918-12924Abstract Full Text PDF PubMed Google Scholar). A Tat mRNA-specific antisense oligonucleotide covalently bound to this fusogenic peptide has been demonstrated to have an increased antiviral activity in vitro, probably as a result of increased cellular uptake (4Bongartz J.P. Aubertin A.-M. Milhaud P.G. Lebleu B. Nucleic Acids Res. 1994; 22: 4681-4688Crossref PubMed Scopus (146) Google Scholar). Peptides adopting an amphipathic conformation at acidic pH largely increased the delivery of plasmid DNA complexed with transferrin-polylysine conjugates (5Wagner E. Plank C. Zatloukal K. Cotten M. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7934-7938Crossref PubMed Scopus (656) Google Scholar). Likewise, amphipathic characteristics have been described for a peptide derived from the third domain of Antennapedia homeodomain (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), which allows the delivery of antisense oligonucleotides or biologically active peptides. Interestingly, this peptide was efficiently translocated through the plasma membrane in the absence of energy (e.g. via a mechanism that does not involve endocytosis). The HIV 1The abbreviations used are: HIV, human immunodeficiency virus; NLS, nuclear localization signal; Boc,t-butyloxycarbonyl; HF, hydrogen fluoride; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; FCS, fetal calf serum; NEM, N-ethylmaleimide; FACS, fluorescence-activated cell sorting; TAMRA-SE, tetramethylrhodamine succinimidyl ester; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. 1The abbreviations used are: HIV, human immunodeficiency virus; NLS, nuclear localization signal; Boc,t-butyloxycarbonyl; HF, hydrogen fluoride; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; FCS, fetal calf serum; NEM, N-ethylmaleimide; FACS, fluorescence-activated cell sorting; TAMRA-SE, tetramethylrhodamine succinimidyl ester; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. Tat transactivation protein is efficiently taken up by cells (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar, 7Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar, 8Vivès E. Charneau P. Van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar), and concentrations as low as 1 nm in the culture media are sufficient to transactivate a reporter gene expressed from the HIV-1 promoter (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar). The domain responsible for this translocation has been ascribed to the region centered on a basic domain of the Tat protein. A peptide extending from residues 37 to 72 allowed the internalization of conjugated proteins such as β-galactosidase or horseradish peroxidase (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). One to two Tat peptides/molecule of protein were sufficient to induce efficient translocation. Likewise, the Tat-(37–62) sequence conjugated to a Fab antibody fragment enhanced its in vitro cell surface association and internalization (10Anderson D.C. Nochols E. Manger R. Woodle D. Barry M. Fritzberg A.R. Biochem. Biophys. Res. Commun. 1993; 194: 876-884Crossref PubMed Scopus (87) Google Scholar). Physicochemical studies involving circular dichroism and energy minimization indicated that the region covering the Tat-(38–49) domain adopted an α-helical structure with amphipathic characteristics (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar). Both biological data and physicochemical studies were in keeping with a crucial role of the α-helix forming domain in Tat uptake. Most of these studies have concerned peptides extending from residues 37 to 72. These include other motifs of interest and in particular a cluster of basic amino acids extending from positions 49 to 58, which does not overlap the presumed amphipathic helical structure. This cluster of basic amino acids (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) appears to be unstructured due to charge repulsions (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar) and contains a nuclear localization signal (NLS) sequence (12Ruben S. Perkins A. Purcell R. Jounc K. Sia R. Burghoff R. Haseltine W.A. Rosen C.A. J. Virol. 1989; 63: 1-8Crossref PubMed Google Scholar).To delineate more precisely which determinants of the Tat-(37–60) peptide are crucial for Tat translocation and nucleolar localization, we have synthesized peptides harboring deletions in the purported α-helix domain or in the basic cluster. These peptides were assayed for their ability to translocate through the cell membrane in several cell lines. Cellular uptake and intracellular distribution were monitored by fluorescence microscopy using peptides labeled with fluorescein maleimide on their C-terminal cysteine or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic domain. Unexpectedly, the α-helix region did not appear to be required for efficient and fast cell uptake. In contrast, the whole basic domain from the Tat peptide appeared necessary for cell internalization.DISCUSSIONSeveral strategies have been proposed to improve the cellular uptake of proteins or nucleic acids. Some of these are based on the use of peptide sequences from proteins known to translocate through the plasma membrane. Along these lines, the HIV-1 Tat protein is able to cross the plasma membrane and to reach the cell nucleus to transactivate the viral genome (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar, 7Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar, 8Vivès E. Charneau P. Van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar). Moreover, a 35-amino acid peptide from Tat is able to promote the intracellular delivery of covalently bound proteins such as β-galactosidase, RNase A, or horseradish peroxidase in several cell lines and tissues (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). This peptide contains a cluster of basic amino acids extending from residues 49 to 58 and a sequence assumed to adopt an α-helical configuration (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar).The present study aimed at delineating whether shorter domains from this Tat peptide would be sufficient for cell internalization. The main determinant required for translocation was identified as the cluster of basic amino acids while the putative α-helix domain appeared dispensable. The full basic domain is required since a peptide deleted from the three arginine residues at its C-terminal end is not taken up by cells, even at high concentration. In keeping with a requirement for the full complement of positive charges in the Tat basic domain, any deletion or substitution of basic charges within the Tat-(48–60) peptide led to a reduced membrane translocating activity. 2E. Vivès, C. Granier, P. Prévot, and B. Lebleu (1997) Lett. Pept. Sci., in press. Shorter peptides such as Tat-(37–58) or Tat-(47–58) were less efficient carriers of proteins than the original 35-amino acid peptide (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). A steric hindrance between such short peptides and the bound protein could have reduced their availability for translocation.Along the same lines, a 16-amino acid peptide from the Antennapedia third helix homeodomain was described as having a good translocation ability through the plasma membrane, and it was initially assumed that its α-helix structure was important (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Likewise, the fusogenic properties of several viral peptides have been ascribed to α-helical determinants (17Gaudin Y. Ruigrok R.W.H. Brunner J. J. Gen. Virol. 1995; 76: 1541-1556Crossref PubMed Scopus (114) Google Scholar). However, it was recently established that the insertion of proline residues, known to disrupt α-helical structures, did not abolish the translocation of the Antennapedia peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). The sequence of this active Antennapedia peptide analogue is Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys. It contains five positive charges (two Arg and three Lys) within a linear sequence of seven residues at its C-terminal end. Similarly, the short Tat basic peptide described here contains eight positive charges within a sequence of nine residues. It is noteworthy that a shorter peptide from Antennapedia deleted of the C-terminal Trp-Lys-Lys residues (i.e. lacking two positive charges) was not internalized (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Although the tryptophan residue was shown to play a role in the translocation event (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), an additional effect of the two Lys residues was not evaluated independently. The data reported with the Antennapedia peptide and those obtained in the present study with the Tat peptide strongly suggest that internalization could be linked to the presence of a high density of positive charges within a short sequence.Both the full-length Antennapedia (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar) and the Tat-(48–60) peptides (Fig. 1) are taken up efficiently at 4 °C. Likewise, several drugs known to interfere with caveolae-mediated uptake did not affect Tat uptake in our studies. Altogether, these studies strongly suggest that endocytosis is not involved in the uptake of these short basic peptides. On the other hand, the incubation of the Tat peptides with various unbound fluorochromes or with a non-permeant peptide did not induce their uptake. Taken together, these experiments do not support the involvement of a significant membrane disruption by Tat basic peptides or a well defined internalization pathway.The mechanism of translocation of the Tat basic peptide could be analogous to the model proposed for the Antennapedia homeodomain peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). A tight ionic interaction between the basic groups of the peptide side chains and the negative charges of the phospholipid heads could induce a local invagination of the plasma membrane. The local reorganization of the phospholipid bilayer would then lead to the formation of inverted micelles with the peptide enclosed in the hydrophilic cavity and ultimately to the cytoplasmic release of the peptide. Because of the presence of a nuclear localization signal, the Tat peptide is rapidly translocated and concentrates in the nucleus. This would limit its release from the cell by the same mechanism. Further experiments are in progress to assess the reality of this working hypothesis. Additional studies will be required to define more accurately the structural requirements for this translocation activity and to uncover the mechanism by which Tat and possibly other basic peptides cross the plasma membrane.The translocation activity of such small Tat-derived peptides is powerful, as nuclear localization was observed after a few minutes of incubation with the cells. Internalization could be monitored in the micromolar concentration range by indirect immunofluorescence with peptide-specific antibodies or even at an order of magnitude lower by direct labeling of the peptide with a fluorochrome. Previous studies with the Antennapedia peptide made use of incubation times of several hours and routine concentrations of 20 μm (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). These differences were confirmed in a comparative study using the same fluorescein-labeling method for both peptides with Antennapedia peptide kindly provided by G. Chassaing and A. Prochiantz (CNRS URA1414, Ecole Normale Supérieure, Paris, France) (data not shown).The indirect immunodetection of the Tat peptide ensures that its ability to translocate through the plasma membrane was not altered by the reporter group itself. In most published studies, the possible influence of the fluorochrome reporter group or of the biotin-linking arm on the behavior of the peptide was not assessed. Along these lines, the biotinylation of a Tat peptide increased by 6-fold its uptake as compared with the non-biotinylated peptide (19Chen L., L. Frankel A.D. Harder J.L. Fawell S. Barsoum J. Anal. Biochem. 1995; 227: 168-175Crossref PubMed Scopus (30) Google Scholar).The internalization properties of these small Tat basic peptides could then be exploited for the intracellular delivery and for the nuclear targeting of conjugated non-permeant molecules. Ongoing work in our group aims at establishing whether the covalent conjugation through various linking arms of short Tat basic peptides to antisense oligonucleotides, to peptide nucleic acids, or to peptides will lead to their nuclear accumulation. Preliminary data indicate that a peptide which did not enter the cell by itself could be efficiently internalized when conjugated to the shorter Tat peptide.2Along the same lines the covalent linking of a 15-mer oligonucleotides to the 16-amino acid Antennapedia peptide led to improved intracellular delivery and to a significant increase in biological activity (20Allinquant B. Hantraye P. Mailleux P. Moya K. Bouillot C. Prochiantz A. J. Cell Biol. 1995; 128: 919-927Crossref PubMed Scopus (234) Google Scholar).Peptides bearing a high density of basic residues might also improve hybridization properties of antisense oligonucleotides. Previous studies have indeed described the enhanced affinities and kinetics of hybridization for its target sequence of an oligonucleotide covalently linked to a polyarginine sequence (21Wei Z. Tung C.H. Zhu T. Dickerhof W.A. Breslauer K.J. Georgopoulos D.E. Leibowitz M.J. Stein S. Nucleic Acids Res. 1996; 24: 655-661Crossref PubMed Scopus (28) Google Scholar). Most "information-rich" molecules, such as oligonucleotides, genes, peptides, or proteins, are poorly taken up by cells since they do not efficiently cross the lipid bilayer of the plasma membrane or of the endocytic vesicles (Ref. 2Lebleu B. Trends Biotechnol. 1996; 14: 109-110Abstract Full Text PDF PubMed Scopus (29) Google Scholar, and references therein). This is considered to be a major limitation for their ex vivo orin vivo use in fundamental studies or in possible clinical applications. These compounds are currently delivered by various techniques including microinjection, electroporation, association with cationic lipids, liposome encapsidation, or receptor-mediated endocytosis. Various problems have been encountered in their use including low transfer efficiency, complex manipulation, cellular toxicity, or immunogenicity, which would preclude their routine usein vivo. As an alternative, several peptides have been successfully used to improve the intracellular delivery of nucleic acids or proteins. The fusogenic properties of influenza virus have been extensively studied in this context. They are currently assigned to a pH-dependent conformational change of the viral hemagglutinin leading to the exposure of its hydrophobic N-terminal region, and to the fusion of the viral and endosomal membranes (3Plank C. Oberhauser B. Mechtler K. Koch C. Wagner E. J. Biol. Chem. 1994; 269: 12918-12924Abstract Full Text PDF PubMed Google Scholar). A Tat mRNA-specific antisense oligonucleotide covalently bound to this fusogenic peptide has been demonstrated to have an increased antiviral activity in vitro, probably as a result of increased cellular uptake (4Bongartz J.P. Aubertin A.-M. Milhaud P.G. Lebleu B. Nucleic Acids Res. 1994; 22: 4681-4688Crossref PubMed Scopus (146) Google Scholar). Peptides adopting an amphipathic conformation at acidic pH largely increased the delivery of plasmid DNA complexed with transferrin-polylysine conjugates (5Wagner E. Plank C. Zatloukal K. Cotten M. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7934-7938Crossref PubMed Scopus (656) Google Scholar). Likewise, amphipathic characteristics have been described for a peptide derived from the third domain of Antennapedia homeodomain (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), which allows the delivery of antisense oligonucleotides or biologically active peptides. Interestingly, this peptide was efficiently translocated through the plasma membrane in the absence of energy (e.g. via a mechanism that does not involve endocytosis). The HIV 1The abbreviations used are: HIV, human immunodeficiency virus; NLS, nuclear localization signal; Boc,t-butyloxycarbonyl; HF, hydrogen fluoride; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; FCS, fetal calf serum; NEM, N-ethylmaleimide; FACS, fluorescence-activated cell sorting; TAMRA-SE, tetramethylrhodamine succinimidyl ester; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. 1The abbreviations used are: HIV, human immunodeficiency virus; NLS, nuclear localization signal; Boc,t-butyloxycarbonyl; HF, hydrogen fluoride; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; FCS, fetal calf serum; NEM, N-ethylmaleimide; FACS, fluorescence-activated cell sorting; TAMRA-SE, tetramethylrhodamine succinimidyl ester; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. Tat transactivation protein is efficiently taken up by cells (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar, 7Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar, 8Vivès E. Charneau P. Van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar), and concentrations as low as 1 nm in the culture media are sufficient to transactivate a reporter gene expressed from the HIV-1 promoter (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar). The domain responsible for this translocation has been ascribed to the region centered on a basic domain of the Tat protein. A peptide extending from residues 37 to 72 allowed the internalization of conjugated proteins such as β-galactosidase or horseradish peroxidase (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). One to two Tat peptides/molecule of protein were sufficient to induce efficient translocation. Likewise, the Tat-(37–62) sequence conjugated to a Fab antibody fragment enhanced its in vitro cell surface association and internalization (10Anderson D.C. Nochols E. Manger R. Woodle D. Barry M. Fritzberg A.R. Biochem. Biophys. Res. Commun. 1993; 194: 876-884Crossref PubMed Scopus (87) Google Scholar). Physicochemical studies involving circular dichroism and energy minimization indicated that the region covering the Tat-(38–49) domain adopted an α-helical structure with amphipathic characteristics (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar). Both biological data and physicochemical studies were in keeping with a crucial role of the α-helix forming domain in Tat uptake. Most of these studies have concerned peptides extending from residues 37 to 72. These include other motifs of interest and in particular a cluster of basic amino acids extending from positions 49 to 58, which does not overlap the presumed amphipathic helical structure. This cluster of basic amino acids (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) appears to be unstructured due to charge repulsions (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar) and contains a nuclear localization signal (NLS) sequence (12Ruben S. Perkins A. Purcell R. Jounc K. Sia R. Burghoff R. Haseltine W.A. Rosen C.A. J. Virol. 1989; 63: 1-8Crossref PubMed Google Scholar). To delineate more precisely which determinants of the Tat-(37–60) peptide are crucial for Tat translocation and nucleolar localization, we have synthesized peptides harboring deletions in the purported α-helix domain or in the basic cluster. These peptides were assayed for their ability to translocate through the cell membrane in several cell lines. Cellular uptake and intracellular distribution were monitored by fluorescence microscopy using peptides labeled with fluorescein maleimide on their C-terminal cysteine or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic domain. Unexpectedly, the α-helix region did not appear to be required for efficient and fast cell uptake. In contrast, the whole basic domain from the Tat peptide appeared necessary for cell internalization. DISCUSSIONSeveral strategies have been proposed to improve the cellular uptake of proteins or nucleic acids. Some of these are based on the use of peptide sequences from proteins known to translocate through the plasma membrane. Along these lines, the HIV-1 Tat protein is able to cross the plasma membrane and to reach the cell nucleus to transactivate the viral genome (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar, 7Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar, 8Vivès E. Charneau P. Van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar). Moreover, a 35-amino acid peptide from Tat is able to promote the intracellular delivery of covalently bound proteins such as β-galactosidase, RNase A, or horseradish peroxidase in several cell lines and tissues (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). This peptide contains a cluster of basic amino acids extending from residues 49 to 58 and a sequence assumed to adopt an α-helical configuration (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar).The present study aimed at delineating whether shorter domains from this Tat peptide would be sufficient for cell internalization. The main determinant required for translocation was identified as the cluster of basic amino acids while the putative α-helix domain appeared dispensable. The full basic domain is required since a peptide deleted from the three arginine residues at its C-terminal end is not taken up by cells, even at high concentration. In keeping with a requirement for the full complement of positive charges in the Tat basic domain, any deletion or substitution of basic charges within the Tat-(48–60) peptide led to a reduced membrane translocating activity. 2E. Vivès, C. Granier, P. Prévot, and B. Lebleu (1997) Lett. Pept. Sci., in press. Shorter peptides such as Tat-(37–58) or Tat-(47–58) were less efficient carriers of proteins than the original 35-amino acid peptide (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). A steric hindrance between such short peptides and the bound protein could have reduced their availability for translocation.Along the same lines, a 16-amino acid peptide from the Antennapedia third helix homeodomain was described as having a good translocation ability through the plasma membrane, and it was initially assumed that its α-helix structure was important (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Likewise, the fusogenic properties of several viral peptides have been ascribed to α-helical determinants (17Gaudin Y. Ruigrok R.W.H. Brunner J. J. Gen. Virol. 1995; 76: 1541-1556Crossref PubMed Scopus (114) Google Scholar). However, it was recently established that the insertion of proline residues, known to disrupt α-helical structures, did not abolish the translocation of the Antennapedia peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). The sequence of this active Antennapedia peptide analogue is Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys. It contains five positive charges (two Arg and three Lys) within a linear sequence of seven residues at its C-terminal end. Similarly, the short Tat basic peptide described here contains eight positive charges within a sequence of nine residues. It is noteworthy that a shorter peptide from Antennapedia deleted of the C-terminal Trp-Lys-Lys residues (i.e. lacking two positive charges) was not internalized (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Although the tryptophan residue was shown to play a role in the translocation event (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), an additional effect of the two Lys residues was not evaluated independently. The data reported with the Antennapedia peptide and those obtained in the present study with the Tat peptide strongly suggest that internalization could be linked to the presence of a high density of positive charges within a short sequence.Both the full-length Antennapedia (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar) and the Tat-(48–60) peptides (Fig. 1) are taken up efficiently at 4 °C. Likewise, several drugs known to interfere with caveolae-mediated uptake did not affect Tat uptake in our studies. Altogether, these studies strongly suggest that endocytosis is not involved in the uptake of these short basic peptides. On the other hand, the incubation of the Tat peptides with various unbound fluorochromes or with a non-permeant peptide did not induce their uptake. Taken together, these experiments do not support the involvement of a significant membrane disruption by Tat basic peptides or a well defined internalization pathway.The mechanism of translocation of the Tat basic peptide could be analogous to the model proposed for the Antennapedia homeodomain peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). A tight ionic interaction between the basic groups of the peptide side chains and the negative charges of the phospholipid heads could induce a local invagination of the plasma membrane. The local reorganization of the phospholipid bilayer would then lead to the formation of inverted micelles with the peptide enclosed in the hydrophilic cavity and ultimately to the cytoplasmic release of the peptide. Because of the presence of a nuclear localization signal, the Tat peptide is rapidly translocated and concentrates in the nucleus. This would limit its release from the cell by the same mechanism. Further experiments are in progress to assess the reality of this working hypothesis. Additional studies will be required to define more accurately the structural requirements for this translocation activity and to uncover the mechanism by which Tat and possibly other basic peptides cross the plasma membrane.The translocation activity of such small Tat-derived peptides is powerful, as nuclear localization was observed after a few minutes of incubation with the cells. Internalization could be monitored in the micromolar concentration range by indirect immunofluorescence with peptide-specific antibodies or even at an order of magnitude lower by direct labeling of the peptide with a fluorochrome. Previous studies with the Antennapedia peptide made use of incubation times of several hours and routine concentrations of 20 μm (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). These differences were confirmed in a comparative study using the same fluorescein-labeling method for both peptides with Antennapedia peptide kindly provided by G. Chassaing and A. Prochiantz (CNRS URA1414, Ecole Normale Supérieure, Paris, France) (data not shown).The indirect immunodetection of the Tat peptide ensures that its ability to translocate through the plasma membrane was not altered by the reporter group itself. In most published studies, the possible influence of the fluorochrome reporter group or of the biotin-linking arm on the behavior of the peptide was not assessed. Along these lines, the biotinylation of a Tat peptide increased by 6-fold its uptake as compared with the non-biotinylated peptide (19Chen L., L. Frankel A.D. Harder J.L. Fawell S. Barsoum J. Anal. Biochem. 1995; 227: 168-175Crossref PubMed Scopus (30) Google Scholar).The internalization properties of these small Tat basic peptides could then be exploited for the intracellular delivery and for the nuclear targeting of conjugated non-permeant molecules. Ongoing work in our group aims at establishing whether the covalent conjugation through various linking arms of short Tat basic peptides to antisense oligonucleotides, to peptide nucleic acids, or to peptides will lead to their nuclear accumulation. Preliminary data indicate that a peptide which did not enter the cell by itself could be efficiently internalized when conjugated to the shorter Tat peptide.2Along the same lines the covalent linking of a 15-mer oligonucleotides to the 16-amino acid Antennapedia peptide led to improved intracellular delivery and to a significant increase in biological activity (20Allinquant B. Hantraye P. Mailleux P. Moya K. Bouillot C. Prochiantz A. J. Cell Biol. 1995; 128: 919-927Crossref PubMed Scopus (234) Google Scholar).Peptides bearing a high density of basic residues might also improve hybridization properties of antisense oligonucleotides. Previous studies have indeed described the enhanced affinities and kinetics of hybridization for its target sequence of an oligonucleotide covalently linked to a polyarginine sequence (21Wei Z. Tung C.H. Zhu T. Dickerhof W.A. Breslauer K.J. Georgopoulos D.E. Leibowitz M.J. Stein S. Nucleic Acids Res. 1996; 24: 655-661Crossref PubMed Scopus (28) Google Scholar). Several strategies have been proposed to improve the cellular uptake of proteins or nucleic acids. Some of these are based on the use of peptide sequences from proteins known to translocate through the plasma membrane. Along these lines, the HIV-1 Tat protein is able to cross the plasma membrane and to reach the cell nucleus to transactivate the viral genome (6Frankel A.D. Pabo C.O. Cell. 1988; 23: 1189-1193Abstract Full Text PDF Scopus (2287) Google Scholar, 7Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (443) Google Scholar, 8Vivès E. Charneau P. Van Rietschoten J. Rochat H. Bahraoui E. J. Virol. 1994; 68: 3343-3353Crossref PubMed Google Scholar). Moreover, a 35-amino acid peptide from Tat is able to promote the intracellular delivery of covalently bound proteins such as β-galactosidase, RNase A, or horseradish peroxidase in several cell lines and tissues (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). This peptide contains a cluster of basic amino acids extending from residues 49 to 58 and a sequence assumed to adopt an α-helical configuration (11Loret E.P. Vivès E. Ho P.S. Rochat H. Van Rietschoten J. Johnson Jr., W.C. Biochemistry. 1991; 30: 6013-6023Crossref PubMed Scopus (60) Google Scholar). The present study aimed at delineating whether shorter domains from this Tat peptide would be sufficient for cell internalization. The main determinant required for translocation was identified as the cluster of basic amino acids while the putative α-helix domain appeared dispensable. The full basic domain is required since a peptide deleted from the three arginine residues at its C-terminal end is not taken up by cells, even at high concentration. In keeping with a requirement for the full complement of positive charges in the Tat basic domain, any deletion or substitution of basic charges within the Tat-(48–60) peptide led to a reduced membrane translocating activity. 2E. Vivès, C. Granier, P. Prévot, and B. Lebleu (1997) Lett. Pept. Sci., in press. Shorter peptides such as Tat-(37–58) or Tat-(47–58) were less efficient carriers of proteins than the original 35-amino acid peptide (9Fawell S. Seery J. Daikh Y. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1096) Google Scholar). A steric hindrance between such short peptides and the bound protein could have reduced their availability for translocation. Along the same lines, a 16-amino acid peptide from the Antennapedia third helix homeodomain was described as having a good translocation ability through the plasma membrane, and it was initially assumed that its α-helix structure was important (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Likewise, the fusogenic properties of several viral peptides have been ascribed to α-helical determinants (17Gaudin Y. Ruigrok R.W.H. Brunner J. J. Gen. Virol. 1995; 76: 1541-1556Crossref PubMed Scopus (114) Google Scholar). However, it was recently established that the insertion of proline residues, known to disrupt α-helical structures, did not abolish the translocation of the Antennapedia peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). The sequence of this active Antennapedia peptide analogue is Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys. It contains five positive charges (two Arg and three Lys) within a linear sequence of seven residues at its C-terminal end. Similarly, the short Tat basic peptide described here contains eight positive charges within a sequence of nine residues. It is noteworthy that a shorter peptide from Antennapedia deleted of the C-terminal Trp-Lys-Lys residues (i.e. lacking two positive charges) was not internalized (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). Although the tryptophan residue was shown to play a role in the translocation event (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar), an additional effect of the two Lys residues was not evaluated independently. The data reported with the Antennapedia peptide and those obtained in the present study with the Tat peptide strongly suggest that internalization could be linked to the presence of a high density of positive charges within a short sequence. Both the full-length Antennapedia (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar) and the Tat-(48–60) peptides (Fig. 1) are taken up efficiently at 4 °C. Likewise, several drugs known to interfere with caveolae-mediated uptake did not affect Tat uptake in our studies. Altogether, these studies strongly suggest that endocytosis is not involved in the uptake of these short basic peptides. On the other hand, the incubation of the Tat peptides with various unbound fluorochromes or with a non-permeant peptide did not induce their uptake. Taken together, these experiments do not support the involvement of a significant membrane disruption by Tat basic peptides or a well defined internalization pathway. The mechanism of translocation of the Tat basic peptide could be analogous to the model proposed for the Antennapedia homeodomain peptide (18Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). A tight ionic interaction between the basic groups of the peptide side chains and the negative charges of the phospholipid heads could induce a local invagination of the plasma membrane. The local reorganization of the phospholipid bilayer would then lead to the formation of inverted micelles with the peptide enclosed in the hydrophilic cavity and ultimately to the cytoplasmic release of the peptide. Because of the presence of a nuclear localization signal, the Tat peptide is rapidly translocated and concentrates in the nucleus. This would limit its release from the cell by the same mechanism. Further experiments are in progress to assess the reality of this working hypothesis. Additional studies will be required to define more accurately the structural requirements for this translocation activity and to uncover the mechanism by which Tat and possibly other basic peptides cross the plasma membrane. The translocation activity of such small Tat-derived peptides is powerful, as nuclear localization was observed after a few minutes of incubation with the cells. Internalization could be monitored in the micromolar concentration range by indirect immunofluorescence with peptide-specific antibodies or even at an order of magnitude lower by direct labeling of the peptide with a fluorochrome. Previous studies with the Antennapedia peptide made use of incubation times of several hours and routine concentrations of 20 μm (1Derossi D. Joliot H.A. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). These differences were confirmed in a comparative study using the same fluorescein-labeling method for both peptides with Antennapedia peptide kindly provided by G. Chassaing and A. Prochiantz (CNRS URA1414, Ecole Normale Supérieure, Paris, France) (data not shown). The indirect immunodetection of the Tat peptide ensures that its ability to translocate through the plasma membrane was not altered by the reporter group itself. In most published studies, the possible influence of the fluorochrome reporter group or of the biotin-linking arm on the behavior of the peptide was not assessed. Along these lines, the biotinylation of a Tat peptide increased by 6-fold its uptake as compared with the non-biotinylated peptide (19Chen L., L. Frankel A.D. Harder J.L. Fawell S. Barsoum J. Anal. Biochem. 1995; 227: 168-175Crossref PubMed Scopus (30) Google Scholar). The internalization properties of these small Tat basic peptides could then be exploited for the intracellular delivery and for the nuclear targeting of conjugated non-permeant molecules. Ongoing work in our group aims at establishing whether the covalent conjugation through various linking arms of short Tat basic peptides to antisense oligonucleotides, to peptide nucleic acids, or to peptides will lead to their nuclear accumulation. Preliminary data indicate that a peptide which did not enter the cell by itself could be efficiently internalized when conjugated to the shorter Tat peptide.2Along the same lines the covalent linking of a 15-mer oligonucleotides to the 16-amino acid Antennapedia peptide led to improved intracellular delivery and to a significant increase in biological activity (20Allinquant B. Hantraye P. Mailleux P. Moya K. Bouillot C. Prochiantz A. J. Cell Biol. 1995; 128: 919-927Crossref PubMed Scopus (234) Google Scholar). Peptides bearing a high density of basic residues might also improve hybridization properties of antisense oligonucleotides. Previous studies have indeed described the enhanced affinities and kinetics of hybridization for its target sequence of an oligonucleotide covalently linked to a polyarginine sequence (21Wei Z. Tung C.H. Zhu T. Dickerhof W.A. Breslauer K.J. Georgopoulos D.E. Leibowitz M.J. Stein S. Nucleic Acids Res. 1996; 24: 655-661Crossref PubMed Scopus (28) Google Scholar). We thank Dr. P. Prévot for fluorescence imaging and computerized analysis of pictures. We are grateful to Drs. J.-P. Briand and J. Neimark (IBMC, Strasbourg, France) as well as to J. Méry (CRBM, Montpellier, France) for the use of their peptide synthesizers and to Dr. C. Granier (U. Montpellier I) for the synthesis of several peptides, to J-A Fehrentz (U. Montpellier I) for HF facilities, to J.-P. Capony (CRBM, CNRS) for amino acid analysis, and to B. Calas (CRBM, CNRS) for electrospray ionization mass spectrometry. We also thank N. Mechti and other colleagues from the Institut de Génétique Moléculaire for fruitful discussions and I. Robbins for proofreading of the manuscript.

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Efficient selection for high-expression transfectants with a novel eukaryotic vector

1991-12-01

Hitoshi Niwa , Ken‐ichi Yamamura , Jun–ichi Miyazaki

Rapid and efficient cosmid cloning

1981-01-01

David Ish‐Horowicz , J. F. Burke

We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, … We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJb8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosporylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1 microgram of target Drosophila DNA gives about 5 x 10(5) clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.

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A microRNA in a Multiple-Turnover RNAi Enzyme Complex

2002-09-19

György Hutvàgner , Phillip D. Zamore

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA … In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells.

Recent advances with liposomes as pharmaceutical carriers

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Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor

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Bin‐Tao Pan , Rose M. Johnstone

Functional siRNAs and miRNAs Exhibit Strand Bias

2003-10-01

Anastasia Khvorova , Angela Reynolds , Sumedha D. Jayasena

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Liposomal drug delivery systems: From concept to clinical applications

2012-10-01

Theresa M. Allen , Pieter R. Cullis

A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation

1986-08-01

Gray D. Shaw , Robert Kamen

Asymmetry in the Assembly of the RNAi Enzyme Complex

2003-10-01

Dianne S. Schwarz , György Hutvàgner , Tingting Du +3 more

In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis

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Dagmar Fischer , Youxin Li , Barbara Ahlemeyer +2 more

Biodegradable nanoparticles for drug and gene delivery to cells and tissue

2003-02-01

Jayanth Panyam , Vinod Labhasetwar

The Drosha-DGCR8 complex in primary microRNA processing

2004-12-01

Jinju Han , Yoontae Lee , Kyu-Hyun Yeom +3 more

RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic … RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs. While Dicer (class III) and other simple RNase III proteins (class I) have been studied intensively, the class II enzyme Drosha remains to be characterized. Here we dissected the action mechanism of human Drosha by generating mutants and by characterizing its new interacting partner, DGCR8. The basic action mechanism of Drosha was found to be similar to that of human Dicer; the RNase III domains A and B form an intramolecular dimer and cleave the 3′ and 5′ strands of the stem, respectively. Human Drosha fractionates at ∼650 kDa, indicating that Drosha functions as a large complex. In this complex, Drosha interacts with DGCR8, which contains two double-stranded RNA (dsRNA)-binding domains. By RNAi and biochemical reconstitution, we show that DGCR8 may be an essential component of the pri-miRNA processing complex, along with Drosha. Based on these results, we propose a model for the action mechanism of class II RNase III proteins.

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Origins and Mechanisms of miRNAs and siRNAs

2009-02-01

Richard W. Carthew , Erik J. Sontheimer

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Argonaute2 Is the Catalytic Engine of Mammalian RNAi

2004-07-30

Jidong Liu , Michelle A. Carmell , Fabiola V. Rivas +6 more

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we … Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

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In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse

1999-09-03

Steven R. Schwarze , Alan L. Ho , Adamina Vocero-Akbani +1 more

Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection … Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

2004-11-01

Jürgen Soutschek , Akin Akinc , Birgit Bramlage +7 more

Intracellular Adenosine Triphosphate (ATP) Concentration: A Switch in the Decision Between Apoptosis and Necrosis

1997-04-21

Marcel Leist , Barbara Single , Anna Federica Castoldi +2 more

Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and … Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) changed from apoptosis to necrosis, when cells were preemptied of adenosine triphosphate (ATP). Nuclear condensation and DNA fragmentation did not occur in cells predepleted of ATP and treated with either of the two inducers, although the kinetics of cell death were unchanged. Selective and graded repletion of the extramitochondrial ATP/pool with glucose prevented necrosis and restored the ability of the cells to undergo apoptosis. Pulsed ATP/depletion/repletion experiments also showed that ATP generation either by glycolysis or by mitochondria was required for the active execution of the final phase of apoptosis, which involves nuclear condensation and DNA degradation.

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Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.

1996-04-01

Margaret A. Goodell , Katja Brose , G Paradis +2 more

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of … Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.

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RNA interference is mediated by 21- and 22-nucleotide RNAs

2001-01-15

Sayda M. Elbashir , Winfried Lendeckel , Thomas Tuschl

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and … Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.

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Rational siRNA design for RNA interference

2004-02-01

Angela Reynolds , Devin Leake , Queta Boese +3 more

mRNA vaccines — a new era in vaccinology

2018-01-12

Norbert Pardi , Michael J. Hogan , Frederick Porter +1 more

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Lipid nanoparticles for mRNA delivery

2021-08-10

Xucheng Hou , Tal Zaks , Róbert Langer +1 more

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RNA interference

2002-07-01

Gregory J. Hannon

Advances in mRNA-LNP lung-targeted delivery strategies

2025-06-26

Xu Liu , Shuhan Ji , Yu‐Dong Cai +3 more | Microstructures

The lung, a vital organ for homeostasis, is vulnerable to various diseases that challenge healthcare systems due to limited treatment options. Fortunately, mRNA-based gene therapy offers a promising solution, demonstrating … The lung, a vital organ for homeostasis, is vulnerable to various diseases that challenge healthcare systems due to limited treatment options. Fortunately, mRNA-based gene therapy offers a promising solution, demonstrating high efficiency and safety across applications in vaccines, protein replacement therapy, and cancer treatment. However, naked mRNA faces challenges like degradation, poor cell penetration, and immunogenicity. The lung’s complex structure further complicates mRNA delivery. In this way, lipid nanoparticles (LNPs) have emerged as an effective solution, demonstrated by their success in COVID-19 mRNA vaccines through superior encapsulation and biocompatibility. Extensive studies focus on developing LNP-based pulmonary mRNA delivery systems for treating viral infections and lung diseases.This review analyzes the current state and developments in mRNA-LNP applications for pulmonary diseases and LNP-based strategies for lung-targeted mRNA delivery. We explore the optimization and development of LNP platforms across four administration routes: nebulized inhalation, intratracheal administration, nasal administration, and systemic administration. Our goal is to provide researchers with a comprehensive reference covering both fundamental principles and cutting-edge developments in pulmonary mRNA-LNP delivery systems.

Harnessing the potential of HSV glycoproteins as cell penetrating peptides through in-silico methods

2025-06-25

Rakesh Ravishankar Rahangdale , Mukesh Pasupuleti , Raghu Chandrashekar Hariharapura | Network Modeling Analysis in Health Informatics and Bioinformatics

Small Interfering RNA (siRNA) for Cardiorenal Disease: Mechanistic Insights from Preclinical and Clinical Studies

2025-06-25

Sukriti Wadehra , Shareen Singh , Sandeep Kaur +2 more | Journal of Cardiovascular Translational Research

Production of Lipid Nanoparticles for mRNA and Vaccines

2025-06-25

Bhupeshwari Patel , Homraj Mahajan , Apoorva Mirche +3 more | CRC Press eBooks

Admixing of mRNA with Pre‐Formed Lipid Nanoparticles Containing a Slightly‐Cationic Ionizable Lipid Allows for Efficient mRNA Transfection In Vitro and In Vivo

2025-06-25

Haixiu Wang , Heleen Lauwers , Mark Gontsarik +5 more | Advanced Healthcare Materials

Abstract Therapeutic mRNA has emerged as a powerful tool in medicine. However, due to its fragility and large size, mRNA requires a carrier for delivery into the cellular cytosol. Lipid … Abstract Therapeutic mRNA has emerged as a powerful tool in medicine. However, due to its fragility and large size, mRNA requires a carrier for delivery into the cellular cytosol. Lipid nanoparticles (LNPs), produced by rapidly mixing an aqueous mRNA solution with an ethanolic solution containing lipids, are currently considered the most advanced carriers for this purpose. Electrostatic interactions between mRNA and the ionizable cationic lipid, combined with hydrophobic interactions among all lipids, lead to self‐assembly into LNPs that accommodate the mRNA in their core. In this study, whether mixing mRNA with pre‐formed, empty LNPs (eLNPs) in an aqueous medium can be a viable alternative for mRNA expression is investigated. It is confirmed that mRNA can associate with eLNPs via electrostatic interactions, with the effectiveness of this association depending on the surface charge of the eLNPs and the ionizable lipid component. Furthermore, post‐loading mRNA into eLNPs demonstrates mRNA expression levels comparable to conventional LNP(mRNA) formulations, both in vitro and in mice. This method of leveraging eLNPs offers a practical alternative to conventional LNP(mRNA) formulation for the rapid screening of multiple mRNAs. It can also enable straightforward use of LNPs for mRNA transfection by users who do not have the capacity to perform LNP formulation.

Harnessing mRNA technology for ischemic heart disease: a review of regenerative and protective therapies

2025-06-24

Matthew Adjmi , Isabelle Tse , Lior Zangi | Cardiology Plus

As ischemic heart disease (IHD) remains the leading cause of mortality worldwide, there is an urgent need for innovative therapies that go beyond symptom management. The irreversible damage to cardiac … As ischemic heart disease (IHD) remains the leading cause of mortality worldwide, there is an urgent need for innovative therapies that go beyond symptom management. The irreversible damage to cardiac tissue following myocardial infarction (MI) and the limited regenerative and proliferative capacity of adult cardiomyocytes (CMs) present significant challenges to the development of treatments capable of restoring cardiac function. This review focuses on emerging modified and non-modified messenger ribonucleic acid (mRNA)-based therapies, which offer targeted and transient protein expression. The studies reviewed here address three major therapeutic strategies: cardiac regeneration, aimed at inducing CM proliferation to restore lost cardiac muscle; cardiac protection, centered on anti-apoptotic and anti-inflammatory methods to mitigate further tissue damage; and cardiovascular regeneration, focused on promoting angiogenesis and restoring vascular integrity after injury. By examining mRNA and modified mRNA (modRNA) therapies across these three approaches, this review showcases mRNA’s promising role in advancing muscular and vascular regenerative and protective therapeutics for IHD.

GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses

2025-06-24

Ahmed M. Albasiony , Amr M. Beltagi , Mohamed E. Khalifa +2 more | International Journal of Molecular Sciences

This study aimed to design dual-responsive chitosan–polylactic acid nanosystems (PLA@CS NPs) for controlled and targeted ledipasvir (LED) delivery to HepG2 liver cancer cells, thereby reducing the systemic toxicity and improving … This study aimed to design dual-responsive chitosan–polylactic acid nanosystems (PLA@CS NPs) for controlled and targeted ledipasvir (LED) delivery to HepG2 liver cancer cells, thereby reducing the systemic toxicity and improving the therapeutic selectivity. Two formulations were developed utilizing ionotropic gelation and w/o/w emulsion techniques: LED@CS NPs with a size of 143 nm, a zeta potential of +43.5 mV, and a loading capacity of 44.1%, and LED-PLA@CS NPs measuring 394 nm, with a zeta potential of +33.3 mV and a loading capacity of 89.3%, with the latter demonstrating significant drug payload capacity. Since most drugs work through interaction with DNA, the in vitro affinity of DNA to LED and its encapsulated forms was assessed using stopped-flow and other approaches. They bind through multi-modal electrostatic and intercalative modes via two reversible processes: a fast complexation followed by a slow isomerization. The overall binding activation parameters for LED (cordination affinity, Ka = 128.4 M−1, Kd = 7.8 × 10−3 M, ΔG = −12.02 kJ mol−1), LED@CS NPs (Ka = 2131 M−1, Kd = 0.47 × 10−3 M, ΔG = −18.98 kJ mol−1) and LED-PLA@CS NPs (Ka = 22026 M−1, Kd = 0.045 × 10−3 M, ΔG = −24.79 kJ mol−1) were obtained with a reactivity ratio of 1/16/170 (LED/LED@CS NPs/LED-PLA@CS NPs). This indicates that encapsulation enhanced the interaction between the DNA and the LED-loaded nanoparticle systems, without changing the mechanism, and formed thermodynamically stable complexes. The drug release kinetics were assessed under tumor-mimetic conditions (pH 5.5, 10 mM GSH) and physiological settings (pH 7.4, 2 μM GSH). The LED@CS NPs and LED-PLA@CS NPs exhibited drug release rates of 88.0% and 73%, respectively, under dual stimuli over 50 h, exceeding the release rates observed under physiological conditions, which were 58% and 54%, thereby indicating that the LED@CS NPs and LED-PLA@CS NPs systems specifically target malignant tissue. Release regulated by Fickian diffusion facilitates tumor-specific payload delivery. Although encapsulation did not enhance the immediate cytotoxicity compared to free LED, as demonstrated by an in vitro cytotoxicity in HepG2 cancer cell lines, it significantly enhanced the therapeutic index (2.1-fold for LED-PLA@CS NPs) by protecting non-cancerous cells. Additionally, the nanoparticles demonstrated broad-spectrum antibacterial effects, suggesting efficacy in the prevention of chemotherapy-related infections. The dual-responsive LED-PLA@CS NPs allowed controlled tumor-targeted LED delivery with better selectivity and lower off-target toxicity, making LED-PLA@CS NPs interesting candidates for repurposing HCV treatments into safer cancer nanomedicines. Furthermore, this thorough analysis offers useful reference information for comprehending the interaction between drugs and DNA.

Role of Tunneling Nanotubes in Arachidonic Acid Transfer and Macrophage Function Reprogramming in Intrahepatic Cholangiocarcinoma

2025-06-24

Mei‐Ru Chen , Shu‐E Zhao , Xiao‐Li Xie +5 more | Advanced Science

Tumor-associated macrophages (TAMs) play a crucial role in tumor progression within the tumor microenvironment (TME) through phenotypic plasticity and functional modulation. While tunneling nanotubes (TNTs) mediate intercellular communication, their role … Tumor-associated macrophages (TAMs) play a crucial role in tumor progression within the tumor microenvironment (TME) through phenotypic plasticity and functional modulation. While tunneling nanotubes (TNTs) mediate intercellular communication, their role in shaping TAMs phenotypes and function remains unclear. This study explores how TNTs facilitate the transfer of tumor-derived materials, particularly fatty acids, to TAMs, affecting macrophage polarization and function. Single-cell RNA sequencing identified heterogeneous macrophage subpopulations in the TME. Enrichment analysis pinpointed key substances transferred via TNTs. Lipidomics and metabolomics analyzed the fatty acids involved. In vitro and in vivo experiments validated TNTs-mediated material transfer, and transcriptomic analysis revealed the associated signaling pathways. TNTs are the primary route for transferring tumor-derived fatty acids, notably arachidonic acid (AA), to macrophages. This transfer reprogrammed TAMs from anti-tumor CD5L+ to pro-tumor TREM2+ phenotypes, increasing CCL18 secretion, reducing phagocytic activity, and impairing CD8+ T cell proliferation. Mechanistically, AA activated the PI3K-AKT pathway, driving TAMs polarization. These findings are confirmed in xenograft models, where TNTs-induced TAMs exhibited enhanced pro-tumor properties. TNTs-mediated transfer of tumor-derived AA reprograms TAMs via PI3K-AKT activation, promoting immune suppression and tumor progression, highlighting TNTs and PI3K-AKT as potential therapeutic targets in iCCA.

Stepwise Administration of Bone-Targeted Lipid Nanoparticles Encapsulating Valproic Acid and TUDCA Facilitates In Vivo Direct Reprogramming for Osteoporosis Treatment

2025-06-24

Hyoeun Park , Woong Jin Cho , Jiseong Kim +7 more | Tissue Engineering and Regenerative Medicine

The rapidly emerging field of RNA therapies for cardiovascular disease

2025-06-24

John Cooke | Cardiology Plus

Boronic acid-modified cell-penetrating peptide exhibits superior performance over natural TAT peptide

2025-06-24

Pritam Ghosh | Frontiers in Chemical Biology

This study explores an enhancement to a cell-penetrating peptide (CPP), specifically cyclic deca arginine (cR10), by modifying it with boronic acid to improve the delivery efficiency of ubiquitin (Ub), an … This study explores an enhancement to a cell-penetrating peptide (CPP), specifically cyclic deca arginine (cR10), by modifying it with boronic acid to improve the delivery efficiency of ubiquitin (Ub), an essential protein that plays various roles in cellular functions. The hypothesis is that adding boronic acid could boost cellular uptake through glycan-boronic acid interactions. This research assesses how the boronic acid-modified cR10 compares to TAT, a natural CPP derived from the HIV-1 transactivator of transcription, in delivering Ub into cells. Experiments with U2OS cells indicated that the boronic acid-linked cR10Ub cargo achieved a fourfold increase in cellular uptake compared to the TAT-Ub conjugate. The findings from this study could contribute to developing new approaches for enhancing protein delivery methods relevant to biomedical research and therapeutic applications.

Characterization of Cisplatin-Resistant Oral Squamous Cell Carcinoma with Enhanced Drug Efflux and Autophagy

2025-06-24

P. Unda | International Journal of Oral and Maxillofacial Surgery

Single‐component cationic polyethylenimine thermogel for sustained and localized gene delivery to combat multi‐drug resistant cancer

2025-06-24

Qianyu Lin , Minting Liu , Lingjie Ke +7 more | BMEMat

Abstract Multi‐drug resistant cancer cells with over‐expression of Bcl2 anti‐apoptotic proteins can be effectively killed by transfecting them with Nur77 transcription factor (pNur77). Previous attempts are generally therapeutically unsatisfactory due … Abstract Multi‐drug resistant cancer cells with over‐expression of Bcl2 anti‐apoptotic proteins can be effectively killed by transfecting them with Nur77 transcription factor (pNur77). Previous attempts are generally therapeutically unsatisfactory due to low sustained gene expression and are further disadvantaged by their multi‐component designs requiring complicated preparation. Herein, we designed a single cationic amphiphilic copolymer from branched poly(ethylenimine) (PEI‐25k), the gold standard non‐viral vector, that functions simultaneously as the non‐viral vector and hydrogel forming polymeric matrix. Our single‐component hydrogel gene delivery platform is highly facile to prepare as it only requires co‐dissolution of the copolymer with pNur77 to form a homogeneous sol. Due to the high cationic charge density of PEI‐25k, this PEI‐based copolymer effectively complexes a high payload of the plasmid. Subsequently, the copolymer's thermogelling ability enables it to spontaneously self‐assemble into a hydrogel depot by simply being warmed to physiological temperatures upon intra‐tumoral injection. Leveraging upon the high transfection efficiency of PEI‐25k, this PEI‐based thermogel achieved prolonged localized release of pNur77 with high transfection efficiency. This leads to successful tumor size reduction and suppressed tumor reoccurrence in mouse models with low systemic cytotoxicity. We believe this single‐component cationic thermogel non‐viral gene delivery platform is highly attractive for gene therapy.

Chiral Supramolecular Clamps for the Passive Cell Diffusion of DNA and siRNA

2025-06-23

Batoul Maatouk , Bappa Maiti , Aliyah Fakim +2 more | Angewandte Chemie

Abstract The safe and effective delivery of DNA and RNA therapeutics continues to be a major hurdle toward personalized medicine, mainly due to the digestion of this genetic material during … Abstract The safe and effective delivery of DNA and RNA therapeutics continues to be a major hurdle toward personalized medicine, mainly due to the digestion of this genetic material during endosomal uptake. Herein, we designed a hydrophobic nonionic urea‐linked molecular clamp (mol‐clamp) based on guanine and phenylalanine (GFUFG) that can readily complex nucleic acids and transport them into cells through passive diffusion. The optimal chiral self‐assembled state was achieved by controlling the dimethyl sulfoxide to water ratio in the solvent mixture. Dictated by the presence of hydrophobic moieties in the mol‐clamp, a secondary structure was formed through intermolecular hydrogen bonding. The negatively charged complex of mol‐clamped nucleic acids was delivered in high efficiency as indicated by the signal of fluorescently labeled ssDNA to both the cytoplasm and the nucleus. The actual efficacy of GFUFG was further evaluated by delivering functional single‐stranded DNA (DNAzyme) and siRNA (si‐BCL‐2), which downregulated EGR‐1 and BCL‐2 by 44% and 53%, respectively. We envisage that designing mol‐clamps that can effectively complex genetic materials and promote passive diffusion rather than endocytosis can improve the field of biologics delivery for individualized therapy and cellular engineering.

Valency-affinity mapping of multivalent liposomes for tunable target cell discrimination

2025-06-23

Víctor García , Paulina Eberts , Brenda M. Ogle +1 more | Drug Delivery

Multivalency can drive high-avidity binding of ligand-functionalized nanoparticles to cells with high target receptor expression, but it can also contribute to off-target binding to low-expression non-target cells. We explored how … Multivalency can drive high-avidity binding of ligand-functionalized nanoparticles to cells with high target receptor expression, but it can also contribute to off-target binding to low-expression non-target cells. We explored how ligand affinity and liposome valency shape the resulting binding performance index (BPI), defined as the product of the proportion of liposome-bound target cells and that of non-bound non-target cells. Designed ankyrin repeat proteins (DARPins) spanning a wide range of HER2-binding affinities were tethered onto PEGylated liposomes at varying concentrations. BPI was initially evaluated in mixed-cell suspensions of HER2high SKBR3 (target) cells and HER2low T47D (non-target) cells, with the highest BPI (> 0.8) observed for high-valency liposomes displaying high-affinity DARPins. To further map the BPI landscape, we measured particle binding to HEK293T cells transiently transfected with HER2-EGFP, leveraging the inherent transfection heterogeneity to generate continuous binding response curves as a function of HER2 expression. HER2high (target) and HER2low (non-target) populations were defined by a HER2 threshold, which was varied across the range of HER2 expression to determine maximum BPI values (> 0.85) and corresponding HER2 threshold optima (HER2OPT). BPI generally tracks with traditional binding selectivity, but BPI is more sensitive to off-target effects or poor on-target binding and thus may better assess particle performance. We further demonstrate that HER2OPT can be rationally increased or decreased by adjusting DARPin valency and affinity (separately or synergistically) to lower or higher values, respectively. The approach outlined here enables rapid testing and optimization of ligand parameters for nanoparticle binding toward a given therapeutic target.

Designing siRNAs against non-structural genes of all serotypes of Dengue virus using RNAi technology – A computational investigation

2025-06-23

Md. Nur Islam , Israt Jahan Asha , Aninda Kumar Gain +6 more | Journal of Genetic Engineering and Biotechnology

Efficient Cytosolic Delivery of siRNA Using Lyophilized and Reconstituted Polymer-siRNA Polyplexes

2025-06-23

Harini Nagaraj , Taewon Jeon , Yağız Anıl Çiçek +4 more | Pharmaceutical Research

On‐Chip De Novo Production of mRNA Vaccine in Lipid Nanoparticles

2025-06-23

Shai Zilberzwige‐Tal , Aviad Levin , Assaf Ezra +7 more | Small

Abstract RNA‐based therapeutics have revolutionized precision medicine due to their unprecedented potency, specificity, and adaptability. However, the inherent limited stability of RNA, including mRNA used in vaccines, is a major … Abstract RNA‐based therapeutics have revolutionized precision medicine due to their unprecedented potency, specificity, and adaptability. However, the inherent limited stability of RNA, including mRNA used in vaccines, is a major obstacle to the full realization of their potential. This instability, coupled with the centralized nature of vaccine production, currently limits the generation of RNA therapeutics at the point of care, which will otherwise fully harness the potential of these agents. Here, a microfluidic platform is presented for on‐demand, personalized synthesis of modified mRNA stabilized by lipid nanoparticles. The design includes trapped biotinylated DNA, tagged T7 RNA polymerase, and a Tesla mixer, allowing the on‐chip synthesis, purification, and encapsulation of mRNA in uniform lipid nanoparticles (LNPs), all conducted seamlessly on the same microfluidic device. This on‐chip microfluidic synthesis approach is found to match standardized mRNA production yields, yet surpasses typical purification methods. Furthermore, as a proof‐of‐concept, the versatility and efficacy of the platform are demonstrated by generating diverse RNA sequences and structures, exhibiting functionality in human cell lines and mouse models. Moreover, an active SARS‐CoV‐2 vaccine is successfully engineered, highlighting the platform's potential for personalized vaccination strategies and offering a promising avenue for high throughput, decentralized vaccine delivery, reduced cold chain dependence, and even advancing current personalized medicine approaches through custom RNA therapeutics.

Integration of polyethylenimine functionalized carbon nanotubes nanocarriers for efficient DNA delivery

2025-06-22

Dong Zeng , Zhiyong He , Jiayu He +6 more | Bioorganic Chemistry

Programmable Comodification of DNA Soccer Framework with Fluorocarbon and Aptamer for Efficient siRNA Delivery

2025-06-22

Luming Zhao , Xiaona Lin , Ni Na Ge +5 more | JACS Au

TRIM5α/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing

2025-06-21

Lijing Wo , Shuhui Qi , Yajun Guo +2 more | Viruses

The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. … The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9TRIM5α−/− without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9TRIM5α−/− displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9TRIM5α−/− and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells.

TfR1-Binding Peptide Conjugation Facilitates Robust and Specific siRNA Delivery to the Central Nervous System

2025-06-21

Tiancheng Fu , Fushun Fan , Yingying Lin +7 more | Bioconjugate Chemistry

Transferrin receptor 1 (TfR1) is a ubiquitously expressed receptor characterized by rapid internalization kinetics and efficient receptor recycling, making it an attractive target for drug delivery. Herein, we investigated the … Transferrin receptor 1 (TfR1) is a ubiquitously expressed receptor characterized by rapid internalization kinetics and efficient receptor recycling, making it an attractive target for drug delivery. Herein, we investigated the potential of TfR1-binding peptide-siRNA conjugates for central nervous system (CNS)-specific gene silencing. A panel of TfR1-binding peptides and conjugation linkers were synthesized to enable siRNA attachment and evaluate their gene-silencing effects. Conjugation with the hTfR No. 894 peptide achieved effective siRNA delivery both in vitro and in vivo. Compared to ribose 2'-O-hexadecyl (C16)-siRNA conjugates, the hTfR No. 894-siRNA conjugation (POC2) elicited favorable pharmacokinetic characteristics and robust and durable silencing of the target gene across CNS regions following local administration, with minimal impact on peripheral tissues. These findings support TfR1-binding peptide conjugation as a promising strategy for CNS-targeted siRNA delivery.

Hydrophobic And Polarized Aromatic Residues Promote Internalization Of Arg‐Rich Cell Penetrating Peptides Through Ionpair‐π Interactions

2025-06-20

Sonia Khemaissa , Antonio Bauzá , Émilie Lesur +4 more | Chemistry - A European Journal

Cell penetrating peptides (CPPs) are small sequences that can cross cell membranes. Arg and Trp are highly prevalent amino‐acids in natural and synthetic efficient CPP sequences. In particular, Trp is … Cell penetrating peptides (CPPs) are small sequences that can cross cell membranes. Arg and Trp are highly prevalent amino‐acids in natural and synthetic efficient CPP sequences. In particular, Trp is essential and cannot be substituted by other hydrophobic or aromatic amino‐acids. The aim of the present study is to decipher the role of Trp in synthetic Arg/Trp CPP sequences. To do so, a small peptide library in which this residue was substituted by other natural or non‐natural amino‐acids was designed. Internalization of these peptides in cells was evaluated and it appeared that combining aromaticity and hydrophobicity in the presence of Arg residues leads to enhanced internalization. The study of the interaction of these peptides with model lipid membranes revealed that the modulation of hydrophobicity promoted insertion in bilayers, but had little impact on the binding affinity. On the other hand, more hydrophobic substitutes of Trp led to more favorable binding enthalpies to heparin. With DFT analysis, we suggest that ion‐pair···π interactions between the aromatic ring and the ion pair formed by the positively charges Arg and the negatively charged cell surface groups can be established, and could be at the origin of the unique internalization properties of Trp‐containing Arg‐rich CPPs.

HMGA2 Attenuates Tendon Stem/Progenitor Cell Senescence Via PI3K/AKT-Mediated Mitophagy Activation and SASP Suppression

2025-06-20

Hao Wang , Guang-Chun Dai , Yucheng Gao +7 more | Stem Cell Reviews and Reports

Endo/Lysosomal-Escapable Lipid Nanoparticle Platforms for Enhancing mRNA Delivery in Cancer Therapy

2025-06-20

Jiapeng Wang , Renjie Chen , Yongyi Xie +3 more | Pharmaceutics

mRNA-based drug development is revolutionizing tumor therapies by enabling precise cancer immunotherapy, tumor suppressor gene restoration, and genome editing. However, the success of mRNA therapies hinges on efficient delivery systems … mRNA-based drug development is revolutionizing tumor therapies by enabling precise cancer immunotherapy, tumor suppressor gene restoration, and genome editing. However, the success of mRNA therapies hinges on efficient delivery systems that can protect mRNA from degradation and facilitate its release into the cytoplasm for translation. Despite the emergence of lipid nanoparticles (LNPs) as a clinically advanced platform for mRNA delivery, the efficiency of endo/lysosomal escape still represents a substantial bottleneck. Here, we summarize the intracellular fate of mRNA-loaded LNPs, focusing on their internalization pathways and processing within the endo-lysosomal system. We also discuss the impact of endo-lysosomal processes on mRNA delivery and explore potential strategies to improve mRNA escape from endo-lysosomal compartments. This review focuses on molecular engineering strategies to enhance LNP-mediated endo/lysosomal escape by optimizing lipid composition, including ionizable lipids, helper lipids, cholesterol, and PEGylated lipids. Additionally, ancillary enhancement strategies such as surface coating and shape management are discussed. By comprehensively integrating mechanistic insights into the journey of LNPs within the endo-lysosome system and recent advances in lipid chemistry, this review offers valuable inspiration for advancing mRNA-based cancer therapies by enabling robust protein expression.

CPPpred-En: Ensemble framework integrating a protein language model and conventional features for highly accurate cell-penetrating peptide prediction

2025-06-20

Yong Eun Jang , Minjun Kwon , Seok Gi Kim +4 more | Computers in Biology and Medicine

Cell-penetrating peptides (CPPs) have gained significant attention for biomedical applications, including drug delivery and therapeutic development, due to their ability to penetrate cell membranes. The accurate prediction of CPPs is … Cell-penetrating peptides (CPPs) have gained significant attention for biomedical applications, including drug delivery and therapeutic development, due to their ability to penetrate cell membranes. The accurate prediction of CPPs is critical for accelerating the design and development of novel peptide-based therapies. Approaches for CPP prediction primarily depend on either peptide characteristic-based conventional features or one or two protein language models (PLMs), but these methods often fail to fully leverage the potential of combining diverse features. To address this limitation, we propose CPPpred-En, a prediction model that evaluates multiple conventional and PLM-based features across various machine learning classifiers, selects high-performing feature-classifier combinations, and integrates them through ensemble learning. The CPPpred-En model, which was trained on both the CPP924 and MLCPP 2.0 datasets, outperformed existing state-of-the-art predictors, achieving an accuracy (Acc) of 97.27 % and a matthews correlation coefficient (MCC) of 0.964 on the CPP924 dataset and an Acc of 96.10 % and an MCC of 0.707 on the MLCPP 2.0 dataset. The ensemble-based strategy demonstrated robustness across different datasets, highlighting the strong ability of the model to generalise. The combination of conventional and PLM features in an ensemble framework is promising approach for improving peptide-based therapeutics. The CPPpred-En model is a highly accurate and reliable tool for the identification of CPPs and their application in drug delivery and targeted therapy.

Systematic analysis of siRNA and mRNA features impacting fully chemically modified siRNA efficacy

2025-06-20

Sarah M. Davis , Samuel Hildebrand , Hannah J. MacMillan +7 more | PubMed

Chemically modified small interfering RNAs (siRNAs) are a promising drug class that silences disease-causing genes via mRNA degradation. Both siRNA-specific features (e.g. sequence, modification pattern, and structure) and target mRNA-specific … Chemically modified small interfering RNAs (siRNAs) are a promising drug class that silences disease-causing genes via mRNA degradation. Both siRNA-specific features (e.g. sequence, modification pattern, and structure) and target mRNA-specific factors contribute to observed efficacy. Systematically defining the relative contributions of siRNA sequence, structure, and modification pattern versus the native context of the target mRNA is necessary to inform design considerations and facilitate the widespread application of this therapeutic platform. To address this, we synthesized a panel of ∼1260 differentially modified siRNAs and evaluated their silencing efficiency against therapeutically relevant mRNAs (APP, BACE1, MAPT, and SNCA) using both reporter-based and native expression assays. Our results demonstrate that the siRNA modification pattern (e.g. level of 2'-O-methyl content) significantly impacts efficacy, while structural features (e.g. symmetric versus asymmetric configurations) do not. Furthermore, we observed substantial differences in the number of effective siRNAs identified per target. These target-specific differences in hit rates are largely mitigated when efficacy is tested in the context of a reporter assay, confirming that native mRNA-specific features influence siRNA performance. Key target-specific factors, including exon usage, polyadenylation site selection, and ribosomal occupancy, partially explained efficacy variability. These insights led to a proposed framework of parameters for optimizing therapeutic siRNA design.

Correction for Smith et al., A twist grain boundary phase in aqueous solutions of the nucleic acid tetramer GTAC

2025-06-20

| Proceedings of the National Academy of Sciences

Lipid-Polymer Hybrid Nanoparticles as a Smart Drug Delivery System for Peptide/Protein Delivery

2025-06-19

Alharith A. A. Hassan , Eslam Ramadan , Katalin Kristó +2 more | Pharmaceutics

The efficient oral delivery of therapeutic proteins and peptides poses a tremendous challenge due to their inherent instability, large molecular size, and susceptibility to enzymatic degradation. Several nanocarrier systems, such … The efficient oral delivery of therapeutic proteins and peptides poses a tremendous challenge due to their inherent instability, large molecular size, and susceptibility to enzymatic degradation. Several nanocarrier systems, such as liposomes, solid lipid nanoparticles, and polymeric nanoparticles, have been explored to overcome these problems. Liposomes and other lipid-based nanocarriers show excellent biocompatibility and the ability to encapsulate hydrophobic and hydrophilic drugs; however, they often suffer from poor structural stability, premature leakage of the loaded drugs, and poor encapsulation efficiency for macromolecular peptides and proteins. On the other hand, polymeric nanoparticles are more stable and allow better control over drug release; nevertheless, they usually lack the necessary biocompatibility and cellular uptake efficiency. Recently, lipid-polymer hybrid nanoparticles (LPHNs) have emerged as an advanced solution combining the structural stability of polymers and the biocompatibility and surface functionalities of lipids to enhance the controlled release, stability, and bioavailability of protein and peptide drugs. In this review, an attempt was made to set a clear definition of the LPHNs and extend the concept and area, so to our knowledge, this is the first review that highlights six categories of the LPHNs based on their anatomy. Moreover, this review offers a detailed analysis of LPHN preparation methods, including conventional and nonconventional one-step and two-step processes, nanoprecipitation, microfluidic mixing, and emulsification methods. Moreover, the material attributes and critical process parameters affecting the output of the preparation methods were illustrated with supporting examples to enable researchers to select the suitable preparation method, excipients, and parameters to be manipulated to get the LPHNs with the predetermined quality. The number of reviews focusing on the formulation of peptide/protein pharmaceutics usually focus on a specific drug like insulin. To our knowledge, this is the first review that generally discusses LPHN-based delivery of biopharmaceuticals. by discussing representative examples of previous reports comparing them to a variety of nanocarrier systems to show the potentiality of the LPHNs to deliver peptides and proteins. Moreover, some ideas and suggestions were proposed by the authors to tackle some of the shortcomings highlighted in these studies. By presenting this comprehensive overview of LPHN preparation strategies and critically analyzing literature studies on this topic and pointing out their strong and weak points, this review has shown the gaps and enlightened avenues for future research.

Viscoelasticity and Its Influence on the Manipulation of <scp>DNA</scp> Molecules

2025-06-19

Xia Wang , Jianjun Dong , Ying Wang +5 more | Microscopy Research and Technique

ABSTRACT The viscoelasticity of DNA molecules in the air were investigated using atomic force microscopy (AFM). For the individual DNA, compared to the randomly‐coiled DNA, the stretched DNA showed smaller … ABSTRACT The viscoelasticity of DNA molecules in the air were investigated using atomic force microscopy (AFM). For the individual DNA, compared to the randomly‐coiled DNA, the stretched DNA showed smaller viscoelasticity, measured in the AFM quantitative imaging (QI) mode. The manipulation of DNA was performed, and it was influenced by its viscoelasticity. Compared to the stretched DNA, significant highlighted accumulations of DNA and lateral dragging tails were more obvious for the randomly‐coiled DNA, which was caused by the elastic recoil and adhesion effects. In addition, the viscoelasticity of DNA aggregates composed of multiple DNA strands were measured. Compared to the individual DNA, they showed larger viscoelasticity. This study serves as a reference for the studies of DNA viscoelasticity‐related behaviors in the air and has potential in gene knockout and targeted gene editing.

Cationic lipopolymer based siRNA delivery for experimental lung cancer treatment

2025-06-19

K Bahadur , Mohammad Nasrullah , Gurveen K. Sandhu +3 more | Biomaterials Advances

Conventional therapeutic approaches often struggle to address "undruggable" or intracellular targets, limiting their effectiveness in treating critical diseases. RNA interference (RNAi), particularly through the delivery of short interfering RNAs (siRNAs), … Conventional therapeutic approaches often struggle to address "undruggable" or intracellular targets, limiting their effectiveness in treating critical diseases. RNA interference (RNAi), particularly through the delivery of short interfering RNAs (siRNAs), has emerged as a promising alternative. In this study, we evaluated the potential of a series of cationic lipopolymers, including ALL-Fect, Leu-Fect, and Prime-Fect, for delivering siRNAs targeting CDC20, Survivin, and STAT5 in lung cancer cell models. These polymers exhibited strong siRNA binding (BC50: 0.17 ± 0.04 to 1.67 ± 0.31) and dissociation (DC50: 57.9 to 13.6 U/mL) properties, forming nanoparticles with ζ-potential of -15 to +23 mV, and particles sizes of 150 to 400 nm suitable for efficient cellular uptake, achieving over 75 % FAM-positive cell populations in lung cancer cells. Remarkably, these complexes demonstrated significant cell killing effects with specific siRNAs even at a low siRNA concentration (20 nM), with maximal effects observed at a polymer/siRNA ratio of 5:1 ratio and 40 nM siRNA concentration, resulting in over 75 % cell killing. The performance of lipid nanoparticles (LNPs) for the delivery of the specific siRNAs was minimal compared to the lipopolymeric carriers under similar conditions. These findings underscore the potential of lipopolymers as safe and effective non-viral vectors for siRNA-based lung cancer therapeutics.

Reversible proliferative arrest induced by rapid depletion of RNase MRP

2025-06-18

Yuan Liu , Shiyang He , Kawon Pyo +7 more | Nature Communications

Modular supramolecular polycations enable efficient delivery of RNA therapeutics and vaccines

2025-06-18

Rafał J. Kopiasz , Hadijatou Sallah , Robert Cavanagh +7 more | Research Square (Research Square)

<title>Abstract</title> We describe a modular, diverse and customisable supramolecular-materials platform which can deliver nucleic acids<italic> in vitro</italic> and <italic>in vivo </italic>with reduced immunogenicity and enhanced stability compared to lipid analogues. … <title>Abstract</title> We describe a modular, diverse and customisable supramolecular-materials platform which can deliver nucleic acids<italic> in vitro</italic> and <italic>in vivo </italic>with reduced immunogenicity and enhanced stability compared to lipid analogues. The chemistries deployed enable the generation of multiple host-guest polycationic structures which can associate with RNA to form polyelectrolyte complexes, but which have the unique feature of reversible cross-links, <italic>via</italic> host-guest interactions of monomers that display aromatic amino acid termini with cucurbit[8]uril (CB[8]). Families of supramolecular polymers can be prepared by simple variation in monomer structure, enabling the tuning of polyelectrolyte complex stability. We demonstrate that these supramolecular polymers and the resultant polyelectrolyte complexes with RNA can be prepared easily via automatable procedures to generate nanoparticles which fully meet Critical Quality Attributes appropriate for manufactured clinical RNA vaccines and therapeutics. We show that these materials deliver RNA to a range of cell types in 2D and 3D culture, displaying reporter-protein expression at levels equivalent to, or greater than, commercial transfection reagents, and with no adverse phenotypic effects <italic>in vitro</italic> or <italic>in vivo</italic>. Finally, we provide diverse evidence for the success of the materials platform across a range of nucleic acid types, with expression of mRNA deep within tumours of an orthotopic Triple-Negative Breast Cancer mouse model, knockdown of a kinase implicated in cancer progression via siRNA and of effective protection by factors of up to1000-fold in terms of reduced viral load in an H1N1 influenza virus challenge model in mice following injections of self-amplifying RNA.

Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift

2025-06-18

Thomas G. Neuman , Riddhi Banik , Surya Karla +5 more | Science Advances

The increasing clinical trials of single-stranded mRNA (ss-mRNA) therapeutics highlight the urgent need to develop efficient, scalable, and economic purification methods. Current diffusion-driven, resin-based purification techniques constrain productivity and rely … The increasing clinical trials of single-stranded mRNA (ss-mRNA) therapeutics highlight the urgent need to develop efficient, scalable, and economic purification methods. Current diffusion-driven, resin-based purification techniques constrain productivity and rely on expensive oligo(dT) ligands for target ss-mRNA poly(A) tail hybridization. To overcome these challenges, we use interfacial molecular forces, such as charge and hydrogen bonds, between nucleic acid variants and a positively charged synthetic microporous membrane to purify ss-mRNA, a desirable therapeutic, from an undesirable impurity, immunogenic double-stranded RNA (dsRNA). Membranes achieved high binding capacities (1.28 mg/m 2 ) and up to 100% ss-mRNA recovery at ~pH 9.0, with optimized surface density (4000 to 10,000 nmol/m 2 ). Purification was operated at rapid flow rates (1.5 ml/min,1000 MV/min) with reusability (>10 trials) and negligible ligand leaching. The key discovery of this cost-effective ligand-less multimodal surface-modified approach is that the addition of the polyamine spermine, which selectively neutralizes dsRNA charge at amine-to-phosphate ratios >450, enhanced separation efficiency.

Brain Targeting Using Nanocolloids for the Management of Multiple Sclerosis

2025-06-18

Smita Jain , Kaisar Raza | Current Drug Delivery

Multiple sclerosis (MS) causes sensory and motor deficiencies by breaking the myelin sheath, which inhibits electrical impulses from reaching affected neurons. The blood-brain barrier (BBB) and unanticipated side effects from … Multiple sclerosis (MS) causes sensory and motor deficiencies by breaking the myelin sheath, which inhibits electrical impulses from reaching affected neurons. The blood-brain barrier (BBB) and unanticipated side effects from inadequate targeting are major hurdles to MS treatment. Nanomedicines are being used to deliver therapeutic chemicals to lesions in order to address the limitations of existing MS therapy approaches. Nano-based therapies with deep BBB penetration and selective targeting have shown promising results, emerging as a possible therapy strategy for MS with improved therapeutic effects. This review will suggest the latest developments in nano-colloidsbased therapy for treating MS by evaluating their advantages and disadvantages.

Innovation in mRNA Vaccines and RNAi via Protein Nanocages

2025-06-18

Sohrab Ahmadivand | Vaccines

Self-assembling protein nanocages (SAPNs) are distinct natural structures formed by the self-assembly of identical subunits, providing a highly efficient platform and a novel strategy for vaccine development and RNAi therapy. … Self-assembling protein nanocages (SAPNs) are distinct natural structures formed by the self-assembly of identical subunits, providing a highly efficient platform and a novel strategy for vaccine development and RNAi therapy. Their internal cavity allows for precise cargo encapsulation, while the externally modifiable surface supports multivalent antigen presentation, thereby enhancing stability, targeted delivery, and immune activation. In addition to serving as stable subunit vaccines with multivalent antigen display, SAPNs can be incorporated into mRNA vaccines (SAPN-RNA vaccines) by pre-fusing with the antigen. This strategy stabilizes secreted antigenic proteins with prolonged presentation to the immune system, and improves vaccine efficacy while reducing off-target effects and minimizing required doses. Additionally, SAPNs can overcome cellular uptake barriers, enhance DNA vaccine efficacy, and enable the co-delivery of antigens and adjuvants. Functionalization with adjuvants or targeting ligands further improves their immunostimulatory properties and specificity. The SAPN-RNAi strategy optimizes siRNA delivery by promoting lysosomal escape, enhancing targeted uptake, and protecting siRNA from degradation through SAPN encapsulation. This review examines the structural and functional properties of protein nanocages and their applications in vaccine design and RNAi delivery, emphasizing their synergistic effects, and exploring current progress, challenges, and future directions. In conclusion, SAPNs represent a versatile multifunctional platform with broad applicability across subunit, mRNA and DNA vaccines, adjuvant co-delivery, and RNAi therapeutics, with significant potential against viral infections.

Increasing polycation hydrophobicity in mRNA polyplex vaccines enhances the efficacy of humoral and cellular immunity induction

2025-06-18

Nan Qiao , Mao Hori , Mitsuru Naito +7 more | Biomaterials

Polycation-based mRNA delivery systems, known as polyplexes, hold significant potential for use in mRNA vaccines and therapeutics. However, their performance has been suboptimal in infectious disease vaccines, which require the … Polycation-based mRNA delivery systems, known as polyplexes, hold significant potential for use in mRNA vaccines and therapeutics. However, their performance has been suboptimal in infectious disease vaccines, which require the induction of both humoral and cellular immunity. Herein, we optimized polycation hydrophobicity to maximize the efficacy of humoral and cellular immunity induction, using biodegradable amphiphilic polyaspartamide derivatives as a platform. The side chains of the polymers contain cationic diethylenetriamine (DET) and hydrophobic 2-cyclohexylethyl (CHE) moieties at varying ratios. Increasing the CHE introduction ratio enhanced immunostimulatory adjuvanticity by activating NLRP3 inflammasome pathway, leading to more efficient activation of cultured dendritic cells. Following subcutaneous injection into mice, polyplexes with higher CHE introduction ratios improved protein expression efficiency in the draining lymph nodes and induced robust germinal center responses. Consequently, antibody responses were enhanced with higher CHE introduction ratios in vaccinations targeting a model antigen and the SARS-CoV-2 spike protein. Furthermore, the vaccination elicited both CD8-positive and CD4-positive T cells in a Th1-skewed manner. In a distribution analysis, protein expression from the delivered mRNA was localized to the injection site and draining lymph nodes, avoiding a safety concern associated with systemic distribution. Collectively, this study demonstrates that the introduction of hydrophobic moieties into polyaspartamide derivatives is an effective and safe strategy to enhance the efficacy of polyplex-based mRNA vaccines targeting infectious diseases.

On the cellular selectivity of targeting peptide-nanoparticle conjugates – a nanoparticle perspective

2025-06-18

Bastian Beitzinger , Bastian Draphoen , Janina Hald +5 more | Expert Opinion on Drug Delivery

Peptide-targeted nanoparticles are promising drug carriers that can enhance drug efficacy at low systemic doses. However, clinical applications are compromised by off-target effects. Nanoparticle surface chemistry fine-tuning is key for … Peptide-targeted nanoparticles are promising drug carriers that can enhance drug efficacy at low systemic doses. However, clinical applications are compromised by off-target effects. Nanoparticle surface chemistry fine-tuning is key for solving these problems. The literature related to peptide-based, active targeting is reviewed, and critically discussed, with focus on the influence of surface chemistry influences on targetability. Furthermore, issues related to limited in vitro-in vivo predictivity are discussed. The potential of more advanced in vitro methods for an increased in vivo prediction is discussed in combination with advanced computational approaches. Efforts toward enhancing endosomal escape are identified as key future developments, in combination with decreasing off-target effects.

Microfluidic synthesis of PEI/plasmid DNA polyplexes for downstream recombinant AAV vector synthesis: Physico-chemical and in vitro transfection study

2025-06-18

Ismail Eş | Chemical Engineering Science

A Novel Copper Ionophore Nanoshuttle (Winged Cu) for Inducing Cuproptosis in B16 Melanoma Cells

2025-06-18

Yuhuan Wu , Ziyao Chang , Wenhao Wang +3 more | Biomolecules

Cuproptosis, a newly discovered copper-dependent programmed cell death pathway, represents a promising approach for anticancer therapy. However, the efficacy of cuproptosis critically depends on intracellular copper accumulation. Traditional copper ionophores … Cuproptosis, a newly discovered copper-dependent programmed cell death pathway, represents a promising approach for anticancer therapy. However, the efficacy of cuproptosis critically depends on intracellular copper accumulation. Traditional copper ionophores have limited therapeutic efficacy due to their reliance on serum copper levels. Therefore, the development of novel copper ionophores to enhance intracellular copper levels is urgently needed. In this study, we targeted a melanoma model and pioneered the application of Bis(2-hydroxyethyl)dithiocarbamic acid copper(II) [Cu(HEDTC)2] as a highly efficient copper ionophore for inducing cuproptosis in B16 melanoma cells. Compared to conventional copper ionophores, Cu(HEDTC)2 exhibits superior intracellular copper delivery efficiency, thereby enhancing the induction of cuproptosis. We further constructed a Cu(HEDTC)2@Soluplus-nanomicelle (CS NM) system designed to disrupt copper ion homeostasis in tumor cells and amplify cuproptosis. In this system, Cu(HEDTC)2, as a novel copper ionophore, significantly enhanced the copper level in B16 melanoma cells. Upon cellular internalization, CS NM underwent degradation and released copper ions, which subsequently triggered cuproptosis by causing abnormal aggregation of mitochondrial lipoylated proteins. This study provides a new experimental foundation and potential therapeutic strategy for cuproptosis-based cancer treatment.

Lipid Nanoparticles Consisting of Sterol-Conjugated Ionizable Lipids Enable Prolonged and Safe mRNA Delivery

2025-06-18

Chenglong Wang , Yiming Zhou , Yanxia Gao +4 more | ACS Applied Materials & Interfaces

mRNA offers a promising therapeutic approach for gene therapy, cancer treatment, and vaccine development. Lipid nanoparticles (LNPs) have emerged as a primary delivery system for mRNA. Ionizable lipids are key … mRNA offers a promising therapeutic approach for gene therapy, cancer treatment, and vaccine development. Lipid nanoparticles (LNPs) have emerged as a primary delivery system for mRNA. Ionizable lipids are key components of LNPs. Given the current limitations of LNPs regarding efficacy, safety, and stability, we undertook this research. We synthesized 11 asymmetric sterol-conjugated ionizable lipids. These lipids and their resulting LNP formulations were screened. Five of these ionizable lipids were selected, and a 40-50% molar ratio was determined to be optimal for the LNP formulation. Luciferase mRNA-loaded LNPs formulated with sterol-conjugated ionizable lipids demonstrated luminescence comparable to that of commercial LNPs composed of ALC-0315 and prolonged protein expression in mice. Furthermore, accelerated stability studies demonstrated that LNPs exhibited good stability. Additionally, LNPs loaded with varicella-zoster virus (VZV) gE mRNA successfully induced both humoral- and T-cell-mediated immune responses. Finally, in vivo safety studies revealed that LNPs based on these ionizable lipids exhibited improved safety profiles compared with commercially available LNPs. In conclusion, these sterol-conjugated ionizable lipid nanoparticles enable prolonged and safe mRNA delivery, representing a significant advance in mRNA delivery technology.

Hydroxychloroquine-Derived Ionizable Lipid Facilitates Spleen-Tropic Transfection and Enhances Cancer Immunotherapy

2025-06-18

Jiaqi Fan , Wei Qi , Pengcheng Yuan +7 more | Chem & Bio Engineering

Exosome-Mediated Mitochondrial Delivery of Antisense Oligonucleotides

2025-06-18

Dora von Trentini , Ivan J. Dmochowski | Nucleic Acid Therapeutics

We present a general method for in-cellulo delivery of 2'-O-methyl (2'-OMe) RNA oligonucleotides (oligos) to mitochondria for antisense applications, with potential for implementation in other mitochondrial DNA (mtDNA)-targeted therapies. Exosomes, … We present a general method for in-cellulo delivery of 2'-O-methyl (2'-OMe) RNA oligonucleotides (oligos) to mitochondria for antisense applications, with potential for implementation in other mitochondrial DNA (mtDNA)-targeted therapies. Exosomes, which are nanoscale, naturally occurring extracellular vesicles (EVs), have been employed for biotechnology applications in oligonucleotide delivery in recent years. We discovered that exosomes from fetal bovine serum (FBS) can be used as a simple and biologically compatible delivery agent of 2'-OMe RNA antisense oligonucleotides to cellular mitochondria, leading to target protein knockdown. While most RNA interference and antisense mechanisms occur in the cytoplasm or nucleus, the need for mitochondrial targeting has become increasingly apparent. Mitochondrial disease describes a variety of currently incurable syndromes that especially affect organs requiring significant energy including the muscles, heart, and brain. Many of these syndromes result from mutations in mtDNA, which codes for the 13 proteins of the oxidative phosphorylation system and are thus often implicated in inherited metabolic disorders.

Cationic Green Solid Lipid nanoparticles by the fatty acid coacervation method for gene delivery to the cornea: preliminary studies on cell and isolated tissue models

2025-06-17

Annalisa Bozza , Marina Beraza-Millor , Julen Rodríguez-Castejón +7 more | European Journal of Pharmaceutics and Biopharmaceutics