Publications by authors named "Philip J Santangelo"

60 Publications

Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts.

PLoS Pathog 2021 05 18;17(5):e1009589. Epub 2021 May 18.

Department of Microbiology, Boston University School of Medicine; National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, United States of America.

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions.
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http://dx.doi.org/10.1371/journal.ppat.1009589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162694PMC
May 2021

The NIH Somatic Cell Genome Editing program.

Nature 2021 04 7;592(7853):195-204. Epub 2021 Apr 7.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.
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http://dx.doi.org/10.1038/s41586-021-03191-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026397PMC
April 2021

Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents.

Nat Biotechnol 2021 06 3;39(6):717-726. Epub 2021 Feb 3.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.
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http://dx.doi.org/10.1038/s41587-021-00822-wDOI Listing
June 2021

Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection.

PLoS Pathog 2020 10 8;16(10):e1008987. Epub 2020 Oct 8.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States of America.

The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.
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http://dx.doi.org/10.1371/journal.ppat.1008987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575074PMC
October 2020

Increased PIP3 activity blocks nanoparticle mRNA delivery.

Sci Adv 2020 Jul 22;6(30):eaba5672. Epub 2020 Jul 22.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.

The biological pathways that affect drug delivery in vivo remain poorly understood. We hypothesized that altering cell metabolism with phosphatidylinositol (3,4,5)-triphosphate (PIP3), a bioactive lipid upstream of the metabolic pathway PI3K (phosphatidylinositol 3-kinase)/AKT/ mTOR (mammalian target of rapamycin) would transiently increase protein translated by nanoparticle-delivered messenger RNA (mRNA) since these pathways increase growth and proliferation. Instead, we found that PIP3 blocked delivery of clinically-relevant lipid nanoparticles (LNPs) across multiple cell types in vitro and in vivo. PIP3-driven reductions in LNP delivery were not caused by toxicity, cell uptake, or endosomal escape. Interestingly, RNA sequencing and metabolomics analyses suggested an increase in basal metabolic rate. Higher transcriptional activity and mitochondrial expansion led us to formulate two competing hypotheses that explain the reductions in LNP-mediated mRNA delivery. First, PIP3 induced consumption of limited cellular resources, "drowning out" exogenously-delivered mRNA. Second, PIP3 triggers a catabolic response that leads to protein degradation and decreased translation.
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http://dx.doi.org/10.1126/sciadv.aba5672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375820PMC
July 2020

Engineering monoclonal antibody-based contraception and multipurpose prevention technologies†.

Biol Reprod 2020 08;103(2):275-285

ZabBio, Inc., San Diego, CA, USA.

Sexually transmitted infections are highly prevalent, and over 40% of pregnancies are unplanned. We are producing new antibody-based multipurpose prevention technology products to address these problems and fill an unmet need in female reproductive health. We used a Nicotiana platform to manufacture monoclonal antibodies against two prevalent sexually transmitted pathogens, HIV-1 and HSV-2, and incorporated them into a vaginal film (MB66) for preclinical and Phase 1 clinical testing. These tests are now complete and indicate that MB66 is effective and safe in women. We are now developing an antisperm monoclonal antibody to add contraceptive efficacy to this product. The antisperm antibody, H6-3C4, originally isolated by Shinzo Isojima from the blood of an infertile woman, recognizes a carbohydrate epitope on CD52g, a glycosylphosphatidylinositol-anchored glycoprotein found in abundance on the surface of human sperm. We engineered the antibody for production in Nicotiana; the new antibody which we call "human contraception antibody," effectively agglutinates sperm at concentrations >10 μg/ml and maintains activity under a variety of physiological conditions. We are currently seeking regulatory approval for a Phase 1 clinical trial, which will include safety and "proof of principle" efficacy endpoints. Concurrently, we are working with new antibody production platforms to bring the costs down, innovative antibody designs that may produce more effective second-generation antibodies, and delivery systems to provide extended protection.
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http://dx.doi.org/10.1093/biolre/ioaa096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401387PMC
August 2020

Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells.

J Neuroinflammation 2020 Jun 20;17(1):197. Epub 2020 Jun 20.

Department of Biomedical Engineering, Duke University, Durham, NC, USA.

Background: Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, "M2-like" macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4).

Methods: To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing.

Results: We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain.

Conclusion: The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.
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http://dx.doi.org/10.1186/s12974-020-01860-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306141PMC
June 2020

Enveloped Virus Inactivation on Personal Protective Equipment by Exposure to Ozone.

medRxiv 2020 May 26. Epub 2020 May 26.

Ozone is a highly oxidizing gas easily generated from atmospheric oxygen with inexpensive equipment and is commonly used for the disinfection of municipal water, foods, and surfaces. We report tests of the ability of ozone to inactivate enveloped respiratory viruses (influenza A virus and respiratory syncytial virus), chosen as more easily handled surrogates for SARS-CoV-2, on N95 respirators and other personal protective equipment (PPE) commonly used in hospitals. At 20 ppm, an ozone concentration easily achieved by standard commercial equipment, the viruses were inactivated with high efficiency as long as the relative humidity was above a threshold value of approximately 50%. In the absence of humidity control, disinfection is more variable and requires considerably longer exposure under relatively dry conditions. This report extends the observations of a previous publication (http://doi.org/10.1080/01919510902747969) to hospital-relevant materials and provides additional details about the relationship of humidity to the antiviral activity of ozone. Home CPAP disinfection devices using ozone can provide effective results for individuals. Ozone did not appear to degrade any of the materials tested except for elastic bands if strained during treatment (such as by the pressure exerted by stapled attachment to N95 respirators). The filtration efficiency of N95 respirator material was not compromised.
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http://dx.doi.org/10.1101/2020.05.23.20111435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273279PMC
May 2020

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

Mol Ther 2020 03 10;28(3):805-819. Epub 2020 Jan 10.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. Electronic address:

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.
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http://dx.doi.org/10.1016/j.ymthe.2020.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054722PMC
March 2020

Mild Innate Immune Activation Overrides Efficient Nanoparticle-Mediated RNA Delivery.

Adv Mater 2020 Jan 19;32(1):e1904905. Epub 2019 Nov 19.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA, 30332, USA.

Clinical mRNA delivery remains challenging, in large part because how physiology alters delivery in vivo remains underexplored. For example, mRNA delivered by lipid nanoparticles (LNPs) is being considered to treat inflammation, but whether inflammation itself changes delivery remains understudied. Relationships between immunity, endocytosis, and mRNA translation lead to hypothesize that toll-like receptor 4 (TLR4) activation reduced LNP-mediated mRNA delivery. Therefore, LNP uptake, endosomal escape, and mRNA translation with and without TLR4 activation are quantified. In vivo DNA barcoding is used to discover a novel LNP that delivers mRNA to Kupffer cells at clinical doses; unlike most LNPs, this LNP does not preferentially target hepatocytes. TLR4 activation blocks mRNA translation in all tested cell types, without reducing LNP uptake; inhibiting TLR4 or its downstream effector protein kinase R improved delivery. The discrepant effects of TLR4 on i) LNP uptake and ii) translation suggests TLR4 activation can "override" LNP targeting, even after mRNA is delivered into target cells. Given near-future clinical trials using mRNA to modulate inflammation, this highlights the need to understand inflammatory signaling in on- and off-target cells. More generally, this suggests an LNP which delivers mRNA to one inflammatory disease may not deliver mRNA to another.
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http://dx.doi.org/10.1002/adma.201904905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029413PMC
January 2020

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways.

Biomaterials 2019 12 21;225:119512. Epub 2019 Sep 21.

Wallace H. Coulter Department of Biomedical Engineering, Georgia; Center for ImmunoEngineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA. Electronic address:

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119512DOI Listing
December 2019

Quantification and Localization of Protein-RNA Interactions in Patient-Derived Archival Tumor Tissue.

Cancer Res 2019 10 3;79(20):5418-5431. Epub 2019 Sep 3.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia.

Abnormal post-transcriptional regulation induced by alterations of mRNA-protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA-protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA-protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA-protein interactions are of interest. SIGNIFICANCE: This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801105PMC
October 2019

Visualization of early events in mRNA vaccine delivery in non-human primates via PET-CT and near-infrared imaging.

Nat Biomed Eng 2019 05 1;3(5):371-380. Epub 2019 Apr 1.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Tech and Emory University, Atlanta, GA, USA.

Visualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals.
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http://dx.doi.org/10.1038/s41551-019-0378-3DOI Listing
May 2019

Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA.

Mol Ther Nucleic Acids 2019 Mar 20;14:52-66. Epub 2018 Nov 20.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Building, Atlanta, GA 30332, USA. Electronic address:

The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
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http://dx.doi.org/10.1016/j.omtn.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304375PMC
March 2019

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo.

J Immunol 2019 01 12;202(2):608-617. Epub 2018 Dec 12.

Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853;

Therapeutic strategies based on in vitro-transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this "proof-of-principle" study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either mRNA or mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.
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http://dx.doi.org/10.4049/jimmunol.1800924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325005PMC
January 2019

Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection.

Nat Commun 2018 10 1;9(1):3999. Epub 2018 Oct 1.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, USA.

The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.
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http://dx.doi.org/10.1038/s41467-018-06508-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167369PMC
October 2018

High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing.

Proc Natl Acad Sci U S A 2018 10 1;115(42):E9944-E9952. Epub 2018 Oct 1.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332;

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.
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http://dx.doi.org/10.1073/pnas.1811276115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196543PMC
October 2018

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4β7.

PLoS Pathog 2018 08 28;14(8):e1007278. Epub 2018 Aug 28.

Section on Intercellular Interactions, Eunice Kennedy-Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States of America.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1007278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130882PMC
August 2018

LEM domain-containing protein 3 antagonizes TGFβ-SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol.

J Biol Chem 2018 10 14;293(41):15867-15886. Epub 2018 Aug 14.

Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908, and

Transforming growth factor-β (TGFβ) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFβ signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFβ, and TGFβ signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFβ through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGFβ signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFβ-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFβ-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFβ by LEMD3, providing a novel target to antagonize pathological TGFβ signaling.
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http://dx.doi.org/10.1074/jbc.RA118.003658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187619PMC
October 2018

In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity.

Bioconjug Chem 2018 09 13;29(9):3072-3083. Epub 2018 Aug 13.

Wallace H. Coulter Department of Biomedical Engineering , Georgia Institute of Technology and Emory University , Krone Engineering Biosystems Building, 950 Atlantic Drive , Atlanta , Georgia 30332 , United States.

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00443DOI Listing
September 2018

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation.

Nano Lett 2018 03 5;18(3):2148-2157. Epub 2018 Mar 5.

Wallace H. Coulter Department of Biomedical Engineering , Georgia Institute of Technology and Emory University School of Medicine , Atlanta , Georgia 30332 , United States.

Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.
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http://dx.doi.org/10.1021/acs.nanolett.8b00432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054134PMC
March 2018

The Role of Integrin αβ in HIV Pathogenesis and Treatment.

Curr HIV/AIDS Rep 2018 04;15(2):127-135

Laboratory of Immunoregulation, National Institutes of Allergy & Infectious Diseases, National Institutes of Health, 10 Center Drive Rm 6A08, Bethesda, MD, 20814, USA.

Purpose Of Review: Acute HIV infection is characterized by high-level viral replication throughout the body's lymphoid system, particularly in gut-associated lymphoid tissues resulting in damage to structural components of gut tissue. This damage is irreversible and believed to contribute to the development of immune deficiencies. Antiretroviral therapy (ART) does not restore gut structure and function. Studies in macaques point to an alternative treatment strategy that may ameliorate gut damage. Integrin αβ mediates the homing of lymphocytes to gut tissues. Vedolizumab, a monoclonal antibody (mAb) antagonist of αβ, has demonstrated efficacy and has been approved for the treatment of inflammatory bowel disease in humans. Here, we describe our current knowledge, and the gaps in our understanding, of the role of αβ in HIV pathogenesis and treatment.

Recent Findings: When administered to macaques prior to infection, a nonhuman primate analogue of vedolizumab prevents transmission of SIV. In combination with ART, this mAb facilitates durable virologic control following treatment interruption. Targeting αβ represents a novel therapeutic approach to prevent and treat HIV infection.
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http://dx.doi.org/10.1007/s11904-018-0382-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882766PMC
April 2018

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction.

Biomaterials 2018 03 6;159:189-203. Epub 2018 Jan 6.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 950 Atlantic Dr. NW, Engineered Biosystems Building, Atlanta, GA, 30332, USA. Electronic address:

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.
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http://dx.doi.org/10.1016/j.biomaterials.2018.01.010DOI Listing
March 2018

RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane.

Nat Commun 2017 09 22;8(1):667. Epub 2017 Sep 22.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, 30332, USA.

The human respiratory syncytial virus G protein plays an important role in the entry and assembly of filamentous virions. Here, we report the use of fluorescently labeled soybean agglutinin to selectively label the respiratory syncytial virus G protein in living cells without disrupting respiratory syncytial virus infectivity or filament formation and allowing for interrogations of respiratory syncytial virus virion assembly. Using this approach, we discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis. This event is then followed by the dynamic formation of filamentous and branched respiratory syncytial virus particles, and assembly with genomic ribonucleoproteins and caveolae-associated vesicles prior to re-insertion into the plasma membrane. We demonstrate that these processes are halted by the disruption of microtubules and inhibition of molecular motors. Collectively, our results show that for respiratory syncytial virus assembly, viral filaments are produced and loaded with genomic RNA prior to insertion into the plasma membrane.Assembly of filamentous RSV particles is incompletely understood due to a lack of techniques suitable for live-cell imaging. Here Vanover et al. use labeled soybean agglutinin to selectively label RSV G protein and show how filamentous RSV assembly, initiated in the cytoplasm, uses G protein recycled from the plasma membrane.
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http://dx.doi.org/10.1038/s41467-017-00732-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610308PMC
September 2017

Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.

Nucleic Acids Res 2017 Jul;45(12):e113

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA 30332, USA.

The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.
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http://dx.doi.org/10.1093/nar/gkx290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499550PMC
July 2017

Arginine-Rich Peptide-Based mRNA Nanocomplexes Efficiently Instigate Cytotoxic T Cell Immunity Dependent on the Amphipathic Organization of the Peptide.

Adv Healthc Mater 2017 Jul 24;6(13). Epub 2017 Apr 24.

Department of Biomedical Molecular Biology, Ghent University, 9052, Ghent, Belgium.

To date, the mRNA delivery field has been heavily dominated by lipid-based systems. Reports on the use of nonlipid carriers for mRNA delivery in contrast are rare in the context of mRNA vaccination. This paper describes the potential of a cell-penetrating peptide containing the amphipathic RALA motif to deliver antigen-encoding mRNA to the immune system. RALA condenses mRNA into nanocomplexes that display acidic pH-dependent membrane disruptive properties. RALA mRNA nanocomplexes enable mRNA escape from endosomes and thereby allow expression of mRNA inside the dendritic cell cytosol. Strikingly, RALA mRNA nanocomplexes containing pseudouridine and 5-methylcytidine modified mRNA elicit potent cytolytic T cell responses against the antigenic mRNA cargo and show superior efficacy in doing so when compared to RALA mRNA nanocomplexes containing unmodified mRNA. RALA's unique sequence and structural organization are vital to act as mRNA vaccine vehicle, as arginine-rich peptide variants that lack the RALA motif show reduced mRNA complexation, impaired cellular uptake and lose the ability to transfect dendritic cells in vitro and to evoke T cell immunity in vivo.
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http://dx.doi.org/10.1002/adhm.201601412DOI Listing
July 2017

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.

Nat Protoc 2017 Jan 15;12(1):150-167. Epub 2016 Dec 15.

Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.
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http://dx.doi.org/10.1038/nprot.2016.168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385890PMC
January 2017

Sustained virologic control in SIV+ macaques after antiretroviral and α4β7 antibody therapy.

Science 2016 10;354(6309):197-202

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the αβ integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4 T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.
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http://dx.doi.org/10.1126/science.aag1276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405455PMC
October 2016

Mirror-enhanced super-resolution microscopy.

Light Sci Appl 2016;5. Epub 2016 Jun 17.

Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia; Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia.

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.
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http://dx.doi.org/10.1038/lsa.2016.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936537PMC
June 2016

DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure.

J Control Release 2016 07 7;234:1-9. Epub 2016 May 7.

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100, USA; Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, GA 30332, USA. Electronic address:

Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes.
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http://dx.doi.org/10.1016/j.jconrel.2016.05.013DOI Listing
July 2016
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