Category Archives: Genetic Engineering

CAMBRIDGE, Mass.--(BUSINESS WIRE)--Kytopen., a transformative biotechnology company offering non-viral delivery that links the discovery, development and manufacturing of engineered cell therapies, today announced it was awarded a SBIR Fast Track grant from the National Institute of Allergy and Infectious Diseases (NIAID), a part of the National Institute of Health (NIH). Kytopen is eligible for up to $2M over the course of the 3-year award as project milestones are successfully completed within the Phase I and Phase II portions of the grant.

Natural killer (NK) cells represent a high impact population for cell therapy, but due to limitations in current methodologies for gene delivery, NK cells remain a largely untapped resource. This SBIR grant will be used to demonstrate that non-viral delivery via Kytopens Flowfect platform can alleviate this limitation on NK cell gene editing at both research and manufacturing scale, which is needed for pre-clinical and clinical studies. Due to the major potential impact NK cells represent in a clinical setting, non-viral Cas Ribonucleoprotein (RNP) gene knockout will allow for novel therapeutic applications in infectious disease, autoimmune disorders, and immuno-oncology.

Paulo Garcia, Kytopens CEO and Co-Founder will serve as the Principal Investigator (PI) on the grant. Dr. Garcia explains that engineered NK cells have tremendous therapeutic promise including the potential to treat solid tumors in an allogeneic modality. The Flowfect platform will facilitate high-throughput target discovery while providing a clear path towards clinical manufacturing of next-generation cell products.

NK cells are a subset of innate immune cells that can respond to threat without antibody priming. This quick response to stimuli makes them an ideal immunotherapy candidate. Yet, genetic modification in NK cells has proven to be difficult using conventional viral and non-viral transfection methodologies. Alternative delivery methods are necessary in order to make genetic modifications at reproducible and efficient rates, while maintaining high cell viability and functionality.

The awarded study leverages continuous fluid flow coupled with low energy electric fields for transfection via a proprietary Flowfect platform (Figure 1). This platform represents a novel approach to non-viral delivery in historically hard-to-transfect human cells. The current research proposes to engineer non-activated NK cells with Cas RNPs for gene editing using the Flowfect platform. To achieve this goal, Kytopen has outlined a two-phase research strategy which focuses on stability and functionality of edited NK cells both in vitro and in vivo.

NIH sponsored grant programs are an integral source of capital for early-stage U.S. small businesses that are creating innovative technologies to improve human health. These programs help small businesses break into the federal research and development arena, create life-saving technologies, and stimulate economic growth. Kytopen is honored to be a recipient of this competitive award from the NIH/NIAID and looks forward to unlocking biological capabilities of engineered NK cells for improving patients lives during the performance of this project.

About the Flowfect Technology

Kytopens proprietary Flowfect platform eliminates the complexity of gene editing and integrates discovery, development and manufacturing in one flexible and scalable non-viral delivery solution. The Flowfect technology utilizes electro-mechanical energy to disrupt the cell membrane and introduce genetic material (such as RNA, DNA, or CRISPR/Cas RNP) to a wide variety of hard-to-transfect primary cells. During the Flowfect process, a solution containing cells and genetic payload suspended in a proprietary buffer flows continuously through a channel while the solution is exposed to a low energy electric field. Due to the continuous flow and low electrical energy required, cells engineered using Flowfect exhibit high viability while also exhibiting high transfection efficiency post-processing. The Flowfect technology utilizes relatively high flow rates enabling cell engineering in minutes for discovery and optimization (e.g. 96 well plate in <10 minutes) and direct scale up to manufacturing volumes of >10mL, engineering over 2 billion cells per minute in a single channel.

About Kytopen

Kytopen, an MIT spin-out, is a transformative biotechnology company that offers a customizable yet scalable multi-solution platform, which seamlessly links the discovery, development and manufacturing phases of cell engineering. Flowfect, a gentle, non-viral delivery method unlocks new therapeutic approaches, by engineering immune cells with minimal disruption, preserving the functionality and viability of human cells and enhancing the cells biology. The Flowfect platform accelerates therapies from the bench to clinical through flexibility and scalability, which drives higher cell yields, faster approvals, and better outcomes from potentially curative cell-based treatments. Kytopens goal is to enable simple and efficient non-viral manufacturing of cell therapies in days versus weeks to increase access to many more patients. For more information, visit: http://www.kytopen.com

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Kytopen Awarded NIH Grant of Up to $2M to Unlock the Power of Engineered Natural Killer (NK) Cells via Flowfect Platform - Business Wire

The report named GlobalGenome Editing or Genome Engineering Market2020 by Company, Regions, Type, and Application, Forecast to 2025 is a broad audit of the market size and patterns with values. The report is a thorough report on worldwide market investigation and experiences. The report is an arrangement of itemized market outline dependent on sorts, application, patterns and openings, consolidations and acquisitions, drivers and restrictions, and a world coming to. The report centers around the arising patterns in the worldwide and provincial spaces on all the huge segments, for example, market limit, cost, value, request and supply, creation, benefit, and serious scene. It offers a board translation of the worldwide Genome Editing or Genome Engineering industry from a scope of data that is gathered through respectable and checked sources.

NOTE:Our experts observing the circumstance across the globe clarifies that the market will create gainful possibilities for makers post COVID-19 emergency. The report means to give an extra outline of the most recent situation, financial stoppage, and COVID-19 effect on the general business.

Market Rundown:

The report investigates past patterns and future possibilities in this report which makes it exceptionally conceivable for the examination of the market. The report gives subtleties of the market by definitions, applications, market plot, item conclusions, and cost structures. The examination moreover shows the market contention scene and a relating point by point examination of the huge dealer/makers in the worldwide Genome Editing or Genome Engineering market. At that point, it presents another endeavor SWOT examination, adventure likelihood, and assessment. The report offers a figure assessment of the valuation of the market 2020-2025.

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The report talks about an entire attitude toward the difficulties existing among the business alongside the rising dangers, imperatives, and constraints. The report contains a full breakdown of the market elements like drivers, development possibilities, item portfolio, mechanical progressions, and a full investigation of the critical contenders of the market. The overall Genome Editing or Genome Engineering market is segmental into types, applications, innovation, end-clients, business verticals, and key geologies.

The central participants concentrated in the report include:Thermo Fisher Scientific Inc (U.S.), Origene Technologies Inc (U.S.), Merck KGAA (Germany), Lonza Group Ltd. (Switzerland), Transposagen Biopharmaceuticals Inc (U.S.), Genscript Biotech Corporation (U.S.), New England Biolabs Inc (U.S.), Sangamo Biosciences Inc (U.S.), Integrated DNA Technologies Inc (U.S.), Horizon Discovery Group Plc (U.K.)

The main sorts of worldwide market items shrouded in this report are:CRISPR, TALEN, ZFN, Antisense, Other Technologies

The most generally utilized downstream fields of the worldwide market canvassed in this report are:Cell Line Engineering, Animal Genetic Engineering, Plant Genetic Engineering, Other Applications

Key areas and nations are canvassed in the worldwide Genome Editing or Genome Engineering market as follows: North America (the United States, Canada, and Mexico), Europe (Germany, France, UK, Russia, and Italy), Asia-Pacific (China, Japan, Korea, India, and Southeast Asia), South America (Brazil, Argentina, and so forth), Middle East and Africa (Saudi Arabia, Egypt, Nigeria, and South Africa)

The report comprises of a summed up market study and in general insights about the business occasions to key partners to grow their business and catch incomes. The worldwide Genome Editing or Genome Engineering market report offers definite examination upheld by solid insights on special and income by players for the time frame 2015-2020. Different subtleties included are organization depiction, significant business, item presentation, ongoing turns of events, deals by area, type, application, and by deals channel.

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Genome Editing or Genome Engineering Market Market: Latest Innovations, Drivers and Industry Key Events 2021 2027 The Courier - The Courier

Washington, June 20, 2021 Databridgemarketresearch.com announces the release of the report Global CRISPR Gene-Editing Market Size, Share & Trends Analysis Report By 2027. Market research report proves to be an ideal solution when it comes to a better understanding of the chemical and materials industry and lead the business growth. Market definitions, segmentation, applications, and value chain structure of this industry are all mentioned in the report. This report provides current as well as upcoming technical and financial details of the industry to 2026. According to this report, the market renovation will mainly take place due to the actions of key players or brands like developments, product launches, joint ventures, mergers, and acquisitions. The company profiles of all the key players and brands that are dominating this Report have been taken into consideration here.

Global CRISPR gene-editing market is rising gradually with a healthy CAGR of 23.35 % in the forecast period of 2019-2026. Growing prevalence of cancer worldwide and expanding the application of CRISPR technology by innovative research from the different academic organizations are the key factors for market growth.

Get a Free Sample Copy of the Report @ (Use Corporate email ID to Get Higher Priority) @https://www.databridgemarketresearch.com/request-a-sample/?dbmr=global-crispr-gene-editing-market

Few of the major competitors currently working in the global CRISPR gene-editing market are Applied StemCell, ACEA BIO, Synthego, Thermo Fisher Scientific Inc, GenScript, Addgene, Merck KGaA, Intellia Therapeutics, Inc, Cellectis, Precision Biosciences, Caribou Biosciences, Inc, Transposagen Biopharmaceuticals, Inc, OriGene Technologies, Inc, Novartis AG, New England Biolabs among others

Global CRISPR Gene-Editing Market By Therapeutic Application (Oncology, Autoimmune/Inflammatory), Application (Genome Engineering, Disease Models, Functional Genomics and Others), Technology (CRISPR/Cas9, Zinc Finger Nucleases and Others), Services (Design Tools, Plasmid and Vector, Cas9 and g-RNA, Delivery System Products and Others), Products (GenCrispr/Cas9 kits, GenCrispr Cas9 Antibodies, GenCrispr Cas9 Enzymes and Others), End-Users (Biotechnology & Pharmaceutical Companies, Academic & Government Research Institutes, Contract Research Organizations and Others), Geography (North America, South America, Europe, Asia-Pacific, Middle East and Africa) Industry Trends and Forecast to 2026

Global CRISPR Gene-Editing Research Methodology

Data Bridge Market Research presents a detailed picture of the market by way of study, synthesis, and summation of data from multiple sources. The data thus presented is comprehensive, reliable, and the result of extensive research, both primary and secondary. The analysts have presented the various facets of the market with a particular focus on identifying the key industry influencers.

Market Drivers

Market Restraints

Some of the Major Highlights of TOC covers:

Chapter 1: Methodology & Scope

Definition and forecast parameters

Methodology and forecast parameters

Data Sources

Chapter 2: Executive Summary

Business trends

Regional trends

Product trends

End-use trends

Chapter 3: CRISPR Gene-Editing Industry Insights

Industry segmentation

Industry landscape

Vendor matrix

Technological and innovation landscape

Chapter 4: CRISPR Gene-Editing Market, By Region

Chapter 5: Company Profile

Business Overview

Financial Data

Product Landscape

Strategic Outlook

SWOT Analysis

Complete report is available (TOC) @https://www.databridgemarketresearch.com/toc/?dbmr=global-crispr-gene-editing-market

Key Developments in the Market:

In April 2019, GenScript has launched Single-stranded DNA Service for CRISPR-based Gene Editing which help the key researchers to have access on the high quality, pure ssDNA for CRISPR-based gene insertion and hence can accelerate the development of gene as well as cell therapy for cancer immunotherapy

In February 2018, Cellectis has received two U.S. patents (US#9,855,297 and US#9,890,393) entiled as Methods for engineering T cells for immunotherapy by using RNA-guided CAS nuclease system for CRISPR Use in T-Cells. The U.S. grant of these patents, the company can generate revenue by out-licensing the products to the pharma companies that are ready to use CRISPR technologies in T-cells

Competitive Analysis:

Global CRISPR gene-editing market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of CRISPR gene-editing market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Thanks for reading this article, you can also get individual chapter wise section or region wise report version like North America, Europe or Asia.

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An absolute way to forecast what future holds is to comprehend the trend today!Data Bridge set forth itself as an unconventional and neoteric Market research and consulting firm with unparalleled level of resilience and integrated approaches. We are determined to unearth the best market opportunities and foster efficient information for your business to thrive in the market. Data Bridge endeavors to provide appropriate solutions to the complex business challenges and initiates an effortless decision-making process.

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Global CRISPR Gene-Editing Market Recent Trends and Developments, Challenges, key drivers and Restraints and Forecast 2021-2028 The Manomet Current -...

Due to unanswered questions into the origins of the coronavirus pandemic, both the U.S. government and scientists have called for a deeper examination into the validity of claims that a virus could have escaped from a lab in Wuhan, China.

Much of the discussion surrounds gain-of-function research. So The Conversation asked David Gillum and Rebecca Moritz, who work closely with virologists on a day-to-day basis to ensure the safety and security of the research, and Sam Weiss Evans and Megan Palmer, who are science and technology policy experts, to explain what this term means and why this kind of research is important.

Any organism can acquire a new ability or property, or gain a function. This can happen through natural selection or a researchers experiments. In research, many different types of experiments generate functions, and some pose certain safety and security concerns.

Scientists use a variety of techniques to modify organisms depending on the properties of the organism itself and the end goal. Some of these methods involve directly making changes at the level of genetic code. Others may involve placing organisms in environments that select for functions linked to genetic changes.

Gain of function can occur in an organism in either nature or the laboratory. Some lab examples include creating more salt- and drought-resistant plants or modifying disease vectors to produce mosquitoes that are resistant to transmitting dengue fever. Gain of function can also be useful for environmental reasons, such as modifying E. coli so that it can convert plastic waste into a valuable commodity.

In the current debate around SARS-CoV-2, the virus that causes COVID-19, gain of function has a much narrower meaning related to a virus becoming easier to move between humans, or becoming more lethal in humans. It is important to remember, though, that the term gain of function by itself covers much more than this type of research.

Two researchers working in a high-containment laboratory hold cell cultures infected with the novel coronavirus.picture alliance via Getty provided by The Conversation,

Gain-of-function experiments may help researchers test scientific theories, develop new technologies and find treatments for infectious diseases. For example, in 2003, when the original SARS-CoV outbreak occurred, researchers developed a method to study the virus in the laboratory. One of the experiments was to grow the virus in mice so they could study it. This work led to a model for researching the virus and testing potential vaccines and treatments.

Gain-of-function research that focuses on potential pandemic pathogens has been supported on the premise that it will help researchers better understand the evolving pathogenic landscape, be better prepared for a pandemic response and develop treatments and countermeasures.

But critics argue that this research to anticipate potential pandemic pathogens does not lead to substantial benefit and is not worth the potential risks. And they say getting out ahead of such threats can be achieved through other means biological research and otherwise. For instance, the current pandemic has provided numerous lessons on the social and behavioral dynamics of disease prevention measures, which could lead to robust new research programs on the cultural aspects of pandemic preparedness. Understanding when the risks of gain-of-function research outweigh the potential benefits and alternatives, therefore, continues to be subject to debate.

Some potential outcomes of gain-of-function research may include the creation of organisms that are more transmissible or more virulent than the original organism or those that evade current detection methods and available treatments. Other examples include engineering organisms that can evade current detection methods and available treatments, or grow in another part of an organism, such as the ability to cross the blood-brain barrier.

There is no such thing as zero risk in conducting experiments. So the question is whether certain gain-of-function research can be performed at an acceptable level of safety and security by utilizing risk-mitigation measures. These strategies for reducing risk include the use of biocontainment facilities, exposure control plans, strict operating procedures and training, incident response planning and much more. These efforts involve dedication and meticulous attention to detail at multiple levels of an institution.

Lab incidents will still occur. A robust biosafety and biosecurity system, along with appropriate institutional response, helps to ensure that these incidents are inconsequential. The challenge is to make sure that any research conducted gain-of-function or otherwise doesnt pose unreasonable risks to researchers, the public and the environment.

Determining whether specific experiments with potential pathogens should be conducted remains a difficult and contentious topic.

There are multiple ways to answer this question. The first is if the research is intended to develop a biological weapon. The United Nations Biological Weapons Convention, which went into effect in 1975, forbids state parties from developing, producing, stockpiling, or otherwise acquiring or sharing biological agents, toxins and equipment that have no justification for peaceful or defensive purposes. There should be no research, then, whether gain-of-function or otherwise, that seeks to purposefully develop a biological weapon.

Another way to answer the question is by focusing on the content of the research, rather than its intent. Through experience, researchers and governments have developed lists of both experiments and organisms that need additional oversight because of their potential safety and security risks. One example of this arose when flu researchers placed a self-imposed pause on gain-of-function research involving the transmissibility of highly pathogenic avian influenza H5N1 viruses in 2012. The U.S. government subsequently imposed a moratorium on the work in 2014. Both moratoriums were lifted by the end of 2017 following a lengthy debate and study of the risks and the development of additional oversight and reporting requirements.

In the past decade, the United States has developed oversight for research that could be directly misused for nefarious purposes. This includes policies on dual-use research of concern (DURC) and policies on pathogens of pandemic potential enhanced to gain transmissibility or virulence.

The main point is that our understanding is constantly evolving. Just before the COVID-19 pandemic began, the U.S. government had started to review and update its policies. It is an open question what lessons will be learned from this pandemic, and how that will reshape our understanding of the value of gain-of-function research. One thing that is likely to happen, though, is that we will rethink the assumptions we have been making about the relationships between biological research, security and society. This may be an opportunity to review and enhance systems of biosecurity and biosafety governance.

David Gillum, Senior Director of Environmental Health and Safety and Chief Safety Officer, Arizona State University and Rebecca Moritz, Biosafety Director and Responsible Official, Colorado State University. This article is republished from The Conversation under a Creative Commons license. Read the original article. Disclosures: David Gillum is the past president of the American Biological Safety Association (ABSA) International. He is a past-judge and member of the safety and security committee for the International Genetically Engineered Machine Competition. Megan J. Palmer receives funding from the Open Philanthropy Project and the Nuclear Threat Initiative. She is on the Council of the Engineering Biology Research Consortium, co-chairs a World Economic Forum Global Future Council on Synthetic Biology, is an Advisor to the International Genetically Engineered Machine Competition, is a member of a World Health Organization Working Group on the Responsible Use of Life Sciences, and is a member of the Board of Directors of Revive and Restore. Sam Weiss Evans receives funding from the Schmidt Futures Foundation. He is a member of the Engineering Biology Research Consortiums Security Working Group, and an Advisor to the international Genetically Engineered Machines Competition. Rebecca Moritz does not work for, consult, own shares in or receive funding from any company or organisation that would benefit from this article, and has disclosed no relevant affiliations beyond their academic appointment.

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Gain Of Function Research And Why It Matters - Science 2.0

Abstract

As numerous diseases are associated with increased local inflammation, directing drugs to the inflamed sites can be a powerful therapeutic strategy. One of the common characteristics of inflamed endothelial cells is the up-regulation of vascular cell adhesion molecule1 (VCAM-1). Here, the specific affinity between very late antigen4 (VLA-4) and VCAM-1 is exploited to produce a biomimetic nanoparticle formulation capable of targeting inflammation. The plasma membrane from cells genetically modified to constitutively express VLA-4 is coated onto polymeric nanoparticle cores, and the resulting cell membranecoated nanoparticles exhibit enhanced affinity to target cells that overexpress VCAM-1 in vitro. A model anti-inflammatory drug, dexamethasone, is encapsulated into the nanoformulation, enabling improved delivery of the payload to inflamed lungs and significant therapeutic efficacy in vivo. Overall, this work leverages the unique advantages of biological membrane coatings to engineer additional targeting specificities using naturally occurring target-ligand interactions.

The chemical and physiological changes associated with inflammation are an important part of the innate immune system (1). Proinflammatory processes can lead to the release of cytokines such as interleukin-6 (IL-6) and tumor necrosis factor, which are capable of effecting vascular changes to improve immune responses at a site of stress or injury (2). These may include vasodilation and an increase in vascular permeability, which can promote more efficient immune cell recruitment (3, 4). On the cellular level, proinflammatory cytokines cause the up-regulation of specific surface markers, including vascular cell adhesion molecule1 (VCAM-1) or intercellular adhesion molecule1 (ICAM-1), which allow for immune cell adhesion at the site of inflammation (5, 6). Although inflammation is an integral process that is required for survival, a dysregulated immune system is implicated in a wide range of disease states (7, 8). The disease relevance of inflammation is further supported by the fact that inflammatory markers such as cellular adhesion molecules are often implicated in pathogenesis (9, 10), and these have been explored as therapeutic and diagnostic targets.

Nanoparticle-based platforms, especially those functionalized with active targeting ligands, have the potential to serve as powerful tools for managing a wide range of diseases associated with inflammation (11). Along these lines, the targeted delivery of anti-inflammatory agents to the vasculature of affected sites via cell adhesion molecules represents a promising strategy (1214). Using inflammation as the cue, a diverse range of nanodelivery systems have been designed to target up-regulated markers such as VCAM-1 and ICAM-1 (1520), and this approach has been leveraged to treat conditions such as cancer and cardiovascular diseases (2123). More recently, cell membrane coating technology has garnered considerable attention in the field of nanomedicine (24, 25). From erythrocytes to cancer cells, virtually any type of cell membrane can be coated onto the surface of nanoparticles, resulting in nanoformulations with enhanced functionality that can be custom-tailored to specific applications (26, 27). In particular, cell membranecoated nanoparticles have proven to be effective drug delivery systems owing to their extended circulation times and disease-homing capabilities (2628). The targeting ability of these biomimetic nanoparticles is often mediated by proteins that are expressed on the source cells, and this bestows the nanoparticles with the ability to specifically interact with various disease substrates. For example, nanoparticles coated with the membrane derived from platelets were shown to specifically target bacteria as well as the exposed subendothelium in damaged vasculature (29). A similar platform was shown to target the lungs in a murine model of cancer metastasis (30). On top of the natural biointerfacing capabilities of cell membranecoated nanoparticles, their traits can be further enhanced by introducing exogenous moieties onto the membrane surface. One way to achieve this is to tether targeting ligands via a lipid anchor, which can then be inserted into the cell membrane (31, 32). Red blood cell membranecoated nanoparticles, which exhibit prolonged blood circulation, have been functionalized in this manner to enhance their cancer targeting ability.

Instead of relying on post-fabrication methods to introduce additional functionality, cell membranecoated nanoparticles can be developed using the membrane from genetically engineered source cells (33). A wide range of tools are available to introduce or up-regulate the expression of specific surface markers (34, 35), and this approach enables researchers to augment the functionality of cell membranebased nanodelivery platforms based on application-specific needs (36, 37). In this study, we genetically engineered cell membranecoated nanoparticles to specifically target sites of inflammation (Fig. 1). Inflamed endothelial cells are known to up-regulate the expression of VCAM-1 to recruit immune cells such as leukocytes that express its cognate ligand, very late antigen4 (VLA-4) (38). To exploit this interaction, we genetically modified a source cell line to stably express VLA-4 and harvested the engineered membrane to coat polymeric nanoparticle cores. A potent anti-inflammatory drug, dexamethasone (DEX), was used as a model payload to be loaded for the treatment of inflammation. The ability of the final nanoformulation to target inflamed cells without compromising the activity of DEX was first tested in vitro. Then, therapeutic efficacy was evaluated in vivo using a murine model of endotoxin-induced lung inflammation.

Wild-type cells were genetically engineered to express VLA-4, which is composed of integrins 4 and 1. Then, the plasma membrane from the genetically engineered cells was collected and coated onto dexamethasone-loaded nanoparticle cores (DEX-NP). The resulting VLA-4expressing cell membranecoated DEX-NP (VLA-DEX-NP) can target VCAM-1 on inflamed lung endothelial cells for enhanced drug delivery.

VLA-4 is a heterodimer that is formed by the association of integrin 4 with integrin 1 (39). To generate a cell line constitutively displaying the full complex, we elected to modify wild-type C1498 cells (C1498-WT), which were confirmed to express high levels of integrin 1 but lack integrin 4 (Fig. 2A). Following viral transduction of C1498-WT to introduce the integrin 4 gene, a subpopulation of the resulting engineered cells (referred to as C1498-VLA) was found to express both VLA-4 components (Fig. 2B). After successfully establishing C1498-VLA, the cells were harvested and their membrane was derived by a process involving cell lysis and differential centrifugation. The cell membrane was then coated onto poly(lactic-co-glycolic acid) (PLGA) nanoparticle cores that were prepared by a single emulsion method. Membrane-coated nanoparticles prepared with the membrane from C1498-WT and C1498-VLA (referred to as WT-NP and VLA-NP, respectively) both had an average diameter of approximately 175 nm, which was slightly larger than the uncoated PLGA cores (Fig. 2C). In terms of zeta potential, the membrane-coated nanoparticles exhibited a surface charge of approximately 20 mV, which was less negative than the PLGA cores (Fig. 2D). Both the size and zeta potential data suggested proper membrane coating, which was further verified for VLA-NP by transmission electron microscopy, which clearly showed a membrane layer surrounding the core (Fig. 2E). Western blotting analysis was used to probe for the two components of VLA-4 on the nanoformulations (Fig. 2F). As expected, both integrins 4 and 1 were found on VLA-NP, whereas only integrin 1 was present on WT-NP. To evaluate long-term stability of the membrane-coated nanoparticles, they were suspended in 10% sucrose solution at 4C, and their size was monitored over the course of 8 weeks (Fig. 2G). Neither nanoparticle sample exhibited a significant increase in size during this period.

(A and B) Expression of integrins 4 and 1 on C1498-WT (A) and C1498-VLA (B) cells was confirmed by flow cytometry. (C and D) The average diameter (C) and surface zeta potential (D) of PLGA cores, WT-NP, and VLA-NP were confirmed by dynamic light scattering (n = 3, mean + SD). (E) Representative transmission electron microscopy image of VLA-NP (scale bar, 100 nm). (F) Western blots for integrins 4 and 1 on WT-NP and VLA-NP. (G) Size of WT-NP and VLA-NP when stored in solution over a period of 8 weeks (n = 3, mean SD).

The binding of VLA-NP was assessed in two different in vitro experiments. First, C1498-WT transduced to constitutively express high amounts of VCAM-1 (referred to as C1498-VCAM) was used as a model target cell. The expression of VCAM-1 on C1498-VCAM was confirmed via flow cytometry (Fig. 3A). Whereas the C1498-WT cells did not show any expression, the C1498-VCAM cells yielded a signal that was over an order of magnitude higher than the isotype control. To evaluate binding, fluorescent dyelabeled WT-NP or VLA-NP were incubated with either C1498-WT or C1498-VCAM (Fig. 3, B and C). For each pairing, the incubation was performed either with or without antiVCAM-1 to block the specific interaction between VLA-4 and VCAM-1. For the samples with blocking, cells were first incubated with the antibody for 30 min before nanoparticle treatment. After incubating with the nanoparticles for 30 min, the cells were washed twice and were analyzed by flow cytometry. The data revealed that there was significant nanoparticle binding only when VLA-NP were paired with C1498-VCAM. The level of binding was reduced back to baseline levels in the presence of antiVCAM-1, thus confirming the specificity of the interaction. In contrast, there was no evidence of specific binding when VLA-NP were paired with C1498-WT, which does not express the cognate receptor for VLA-4. The same held true for the WT-NP paired with either cell type, where antibody blocking had no impact on the relative nanoparticle binding.

(A) Expression of VCAM-1 on C1498-WT and C1498-VCAM cells (gray, isotype antibody; green, antiVCAM-1). (B and C) Binding of WT-NP (B) or VLA-NP (C) to C1498-WT or C1498-VCAM cells; blocking was performed by preincubating cells with antiVCAM-1 (n = 3, mean + SD). ****P < 0.0001, Students t test. (D) Expression of VCAM-1 on untreated or LPS-treated bEnd.3 cells (gray, isotype antibody; green, antiVCAM-1). (E and F) Binding of WT-NP (E) or VLA-NP (F) to untreated or LPS-treated bEnd.3 cells; blocking was performed by preincubating cells with antiVCAM-1 (n = 3, mean + SD). **P < 0.01, Students t test.

Next, we elected to study the nanoparticle binding to endothelial cells, which represent a more biologically relevant target compared to the artificially engineered C1498-VCAM cells. For this purpose, we used a murine brain endothelial cell line, bEnd.3, whose VCAM-1 expression can be up-regulated in the presence of proinflammatory signals (40). To induce an inflamed state, bEnd.3 cells were treated with bacterial lipopolysaccharide (LPS), and the level of VCAM-1 expression was evaluated using flow cytometry (Fig. 3D). Whereas expression of VCAM-1 was near baseline levels for the untreated bEnd.3 cells, those that were treated with LPS exhibited a distinct population with elevated VCAM-1. As we observed in the previous experiment with C1498-VCAM cells, enhanced nanoparticle binding was only observed when VLA-NP were paired with inflamed bEnd.3 cells, and antibody blocking reduced the levels back to baseline (Fig. 3, E and F). When incubating with noninflamed bEnd.3 cells, there was no evidence of specific binding interactions, and the same held true for the control WT-NP paired with bEnd.3 cells regardless of their inflammatory status. The data in these two studies confirmed the successful engineering of membrane-coated nanoparticles with the ability to target inflammation based on the interaction between VLA-4 and VCAM-1.

As a model anti-inflammatory payload, we selected DEX, which was loaded into the PLGA core by a single emulsion method before coating with either C1498-WT or C1498-VLA membrane to yield DEX-loaded WT-NP or VLA-NP (referred to as WT-DEX-NP or VLA-DEX-NP, respectively). When the drug content was measured by high-performance liquid chromatography (HPLC), it was determined that the encapsulation efficiency and drug loading yield were approximately 11 and 2 weight % (wt %), respectively (Fig. 4A). To evaluate drug release, VLA-DEX-NP was dialyzed against a large volume of phosphate-buffered saline (PBS), and the amount of drug retained within the nanoparticles was quantified over time (Fig. 4B). The results revealed an initial burst, where approximately 80% of the drug payload was released in the first hour, followed by a sustained release. The release profile was in agreement with previous reports on DEX-loaded PLGA formulations (41, 42), and the data showed a good fit with the Peppas-Sahlin model with a regression coefficient of 0.978 (43). To evaluate the biological activity of the DEX loaded within the nanoparticles, we used an in vitro assay based on the LPS treatment of DC2.4 dendritic cells, which causes an elevation in the levels of proinflammatory cytokines such as IL-6 (Fig. 4C). DC2.4 cells were first treated with either free DEX or VLA-DEX-NP for 2 hours, followed by incubation with LPS overnight. The supernatant was then collected to measure the concentration of IL-6 by an enzyme-linked immunosorbent assay (ELISA). It was shown that both free DEX and VLA-DEX-NP were able to attenuate IL-6 secretion in a drug concentrationdependent manner (Fig. 4D). Although free DEX more efficiently lowered IL-6 levels at drug concentrations of 0.01 and 0.1 M, the level of inflammation was reduced to levels near baseline for both free DEX and VLA-DEX-NP at 1 M of drug. The data indicated that the activity of the drug payload was retained after being loaded inside of VLA-NP. It was confirmed that neither PLGA cores nor VLA-NP without DEX loading had an impact on the level of IL-6 production by the DC2.4 cells (Fig. 4E).

(A) Drug loading (DL) and encapsulation efficiency (EE) of dexamethasone (DEX) into VLA-NP (n = 3, mean + SD). (B) Drug release profile of VLA-DEX-NP (n = 3, mean SD). The data were fitted using the Peppas-Sahlin equation (dashed line). (C) Secretion of IL-6 by LPS-treated DC2.4 cells (n = 3, mean + SD). UD, undetectable. (D) Secretion of IL-6 by LPS-treated DC2.4 cells preincubated with DEX in free form or loaded into VLA-NP (n = 3, mean SD). (E) Relative inflammatory response, as measured by IL-6 secretion, of DC2.4 cells treated with LPS only, LPS and PLGA nanoparticles, LPS and VLA-NP, PLGA nanoparticles only, or VLA-NP only; all of the nanoparticles were empty without DEX loading (n = 3, mean + SD). NS, not significant (compared to the LPS-only group), one-way analysis of variance (ANOVA).

After confirming the biological activity of the VLA-DEX-NP formulation in vitro, we next sought to evaluate the formulation in vivo using a murine model of lung inflammation. The model was established by intratracheal injection of LPS directly into the lungs of BALB/c mice. To evaluate targeting ability, fluorescently labeled WT-NP or VLA-NP were injected intravenously after the induction of lung inflammation. After 6 hours, major organs, including the heart, lungs, liver, spleen, kidneys, and blood, were collected to assess nanoparticle biodistribution (Fig. 5A). The majority of the nanoparticles accumulated in the liver and spleen. Notably, a significant increase in accumulation of VLA-NP was observed in the lungs compared to WT-NP. This in vivo targeting result was in agreement with the in vitro findings where VLA-NP were able to specifically bind to inflamed cells. The safety of the formulation was assessed by monitoring the plasma levels of creatinine, a marker of kidney toxicity that was previously studied in the context of DEX nanodelivery (44). After 9 days of repeated daily administrations of free DEX or VLA-DEX-NP into healthy mice, it was shown that the creatinine concentration in mice receiving VLA-DEX-NP remained consistent with baseline levels, whereas it was significantly elevated in mice administered with free DEX (Fig. 5B).

(A) Biodistribution of WT-NP or VLA-NP in a lung inflammation model 6 hours after intravenous administration (n = 3, mean + SD). *P < 0.05, Students t test. AU, arbitrary units. (B) Creatinine levels in the plasma of mice after repeated daily administrations for 9 days with free DEX or VLA-DEX-NP (n = 3, mean + SD). *P < 0.05, one-way ANOVA. (C) IL-6 levels in the lung tissue of mice intratracheally challenged with LPS and then treated intravenously with vehicle solution, free DEX, WT-DEX-NP, or VLA-DEX-NP (n = 3, mean SD). ***P < 0.001, ****P < 0.0001 (compared to VLA-DEX-NP), one-way ANOVA. (D) Representative hematoxylin and eosinstained lung histology sections of mice intratracheally challenged with LPS and then treated intravenously with vehicle solution, free DEX, WT-DEX-NP, or VLA-DEX-NP (scale bar, 100 m).

The therapeutic efficacy of VLA-DEX-NP was then evaluated following the same experimental design as the targeting study. After 6 hours, the lungs were collected and homogenized, and the homogenate was then clarified by centrifugation and filtered through a 0.22-m porous membrane before measuring the concentration of IL-6 by ELISA. As shown in Fig. 5C, the VLA-DEX-NP formulation was able to completely abrogate lung inflammation, while both free DEX and WT-DEX-NP did not have any discernable effect. The fact that WT-DEX-NP were not able to significantly reduce lung IL-6 levels suggested that systemic exposure to DEX was not a major contributor to the efficacy observed with VLA-DEX-NP. The efficacy of the formulation against lung inflammation was further confirmed by analyzing lung sections stained with hematoxylin and eosin (Fig. 5D). Leukocyte recruitment and peribronchial thickening, which are hallmarks of lung inflammation (45, 46), were prominent in the lungs of mice receiving no treatment, free DEX, or WT-DEX-NP. In contrast, minimal leukocyte recruitment and no peribronchial thickening were observed for the group treated with VLA-DEX-NP, and there were no other signs of toxicity present in these lung sections. Overall, the results from the in vivo studies confirmed the benefit of targeted delivery to inflamed lungs using VLA-NP as a drug nanocarrier.

In conclusion, we have engineered cell membranecoated nanoparticles that can be used to specifically target and treat localized lung inflammation via systemic administration. A host cell positive for integrin 1 was modified to express integrin 4. Together, the two protein markers formed VLA-4, which specifically interacts with VCAM-1, a common marker for inflammation found on vascular endothelia. Nanoparticles fabricated using the membrane from these genetically engineered cells were able to leverage this natural affinity to target inflamed sites, including in a murine model of LPS-induced lung inflammation. When the nanoparticles were loaded with DEX, an anti-inflammatory drug, significant therapeutic efficacy was achieved in vivo. Future studies will comprehensively evaluate the safety profile of the VLA-DEX-NP formulation, obtain additional lung-specific efficacy readouts, elucidate the optimal time window for treatment, and assess clinical relevance using additional animal models of severe inflammatory disease. As pathological inflammation is heavily implicated in a number of important disease conditions (7, 47), the reported biomimetic platform could be leveraged to improve the in vivo activity of various therapeutic payloads through enhanced targeting. Notably, VCAM-1 up-regulation has been observed in renal pathologies as well as in inflamed cerebral vasculature (48, 49). In addition, DEX has been shown to be effective at managing the inflammation associated with COVID-19 (50), and a targeted formulation capable of localizing the drug to the lungs may help to further boost its therapeutic profile. In this work, we specifically engineered the nanoparticles to display VLA-4, which is a complex, multicomponent membranebound ligand that would otherwise be infeasible to incorporate using traditional synthetic strategies. This highlights the advantages of using genetic engineering techniques to expand the wide-ranging utility of cell membrane coating technology. In particular, the generalized application of this approach would enable researchers to streamline the development of new targeted nanoformulations by using target-ligand interactions that occur in nature. Combined with the biocompatibility and biointerfacing characteristics that are inherent to cell membrane coatings, the work presented here could initiate a new wave of biomimetic nanomedicine with finely crafted functionalities.

Wild-type C1498 mouse leukemia cells (TIB-49, American Type Culture Collection) were cultured at 37C in 5% CO2 with Dulbeccos modified Eagles medium [DMEM; with l-glutamine, glucose (4.5 g/liter), and sodium pyruvate; Corning] supplemented with 10% bovine growth serum (BGS; Hyclone) and 1% penicillin-streptomycin (Pen-Strep; Gibco). Engineered C1498-VCAM cells were cultured with DMEM supplemented with 10% U.S. Department of Agriculture (USDA) fetal bovine serum (FBS; Omega Scientific), 1% Pen-Strep, and hygromycin B (400 g/ml; InvivoGen). Engineered C1498-VLA cells were cultured with DMEM supplemented with 10% USDA FBS, 1% Pen-Strep, and puromycin (1 g/ml; InvivoGen). bEnd.3 mouse brain endothelial cells (CRL-2299, American Type Culture Collection) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep. AmphoPhoenix cells (obtained from the National Gene Vector Biorepository) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep. DC2.4 mouse dendritic cells (SCC142, Sigma-Aldrich) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep.

Engineered C1498-VLA and C1498-VCAM cells were created by transducing C1498-WT. Briefly, the genes for integrin 4 (MG50049-M, Sino Biological) and VCAM-1 (MG50163-UT, Sino Biological) gene were cloned into pQCXIP and pQCXIH plasmids (Clontech), respectively, using an In-Fusion HD cloning kit (Clontech) following the manufacturers protocol, yielding pQCXIP-4 and pQCXIH-VCAM-1. AmphoPhoenix cells were plated onto 100-mm tissue culture dishes containing 10 ml of medium at 3 105 cells/ml and cultured overnight. The cells were transfected with pQCXIP-4 or pQCXIH-VCAM-1 using Lipofectamine 2000 (Invitrogen) following the manufacturers instructions. The supernatant of the transfected AmphoPhoenix was collected and used to resuspend C1498-WT cells, which were then centrifuged at 800g for 90 min. After the spin, the transduced cells were incubated for 4 hours before the media were changed with fresh media. Fluorescently labeled antibodies, including FITC (fluorescein isothiocyanate) anti-mouse CD49d (R1-2, BioLegend), Alexa647 anti-mouse/rat CD29 (HM1-1, BioLegend), or PE (phycoerythrin) anti-mouse CD106 (STA, BioLegend), were used to assess the expression levels of VLA-4 or VCAM-1. Data were collected using a Becton Dickinson FACSCanto-II flow cytometer and analyzed using FlowJo software. All of the engineered cells were sorted using a Becton Dickinson FACSAria-II flow cytometer to select for cells expressing high levels of VLA-4 or VCAM-1.

The membranes from C1498-WT and engineered C1498-VLA cells were derived using a previously described method with some modifications (51). First, the cells were harvested and washed in a starting buffer containing 30 mM tris-HCl (pH 7.0) (Quality Biological) with 0.0759 M sucrose (Sigma-Aldrich) and 0.225 M d-mannitol (Sigma-Aldrich). The washed cells were resuspended in an isolation buffer containing 0.5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (Sigma-Aldrich), a phosphatase inhibitor cocktail (Sigma-Aldrich), and a protease inhibitor cocktail (Sigma-Aldrich). Then, the cells were homogenized using a Kinematica Polytron PT 10/35 probe homogenizer at 70% power for 15 passes. The homogenate was first centrifuged at 10,000g in a Beckman Coulter Optima XPN-80 ultracentrifuge for 25 min. The supernatant was then collected and centrifuged at 150,000g for 35 min. The resulting pellet of cell membrane was washed and stored in a solution containing 0.2 mM ethylenediaminetetraacetic acid (USB Corporation) in UltraPure DNase-free/RNase-free distilled water (Invitrogen). Total membrane protein content was quantified by a BCA protein assay kit (Pierce).

Polymeric cores were prepared by a single emulsion process using carboxyl-terminated 50:50 PLGA (0.66 dl/g; LACTEL absorbable polymers). For DEX-loaded PLGA cores, 500 l of PLGA (50 mg/ml) in dichloromethane (DCM; Sigma-Aldrich) was mixed with 500 l of DEX (10 mg/ml) in acetone. This mixture was added to 5 ml of 10 mM tris-HCl (pH 8) and sonicated using a Thermo Fisher Scientific 150E Sonic Dismembrator at 70% power for 2 min. The sonicated mixture was added to 10 ml of 10 mM tris-HCl (pH 8) and was magnetically stirred at 700 rpm overnight. For 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD, ex/em = 644/663 nm; Biotium) labeling, 500 l of PLGA (50 mg/ml) in DCM was mixed with 500 l of DiD (20 g/ml) in DCM. This mixture was added to 5 ml of 10 mM tris-HCl (pH 8) and sonicated using a Thermo Fisher Scientific 150E Sonic Dismembrator at 70% power for 2 min. The sonicated mixture was added to 10 ml of 10 mM tris-HCl (pH 8) and was magnetically stirred at 700g for 3 hours. Empty PLGA core preparation followed the same procedure, except substituting the DiD solution for 500 l of neat DCM. To coat the polymeric cores with cell membranes, the nanoparticle cores were first centrifuged at 21,100g for 8 min. The pellets were resuspended in solution containing membranes derived from C1498-WT or C1498-VLA. The mixture was sonicated in a 1.5-ml disposable sizing cuvette (BrandTech Scientific Inc.) using a Thermo Fisher Scientific FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 3 min. For the in vitro studies, UltraPure water and sucrose were added to adjust the polymer concentration to 1 mg/ml and the sucrose concentration to 10%. For the in vivo studies, UltraPure water and sucrose were added to adjust the polymer concentration to 10 mg/ml and the sucrose concentration to 10%.

The size and surface zeta potential of WT-NP and VLA-NP were measured by dynamic light scattering using a Malvern ZEN 3600 Zetasizer. For electron microscopy visualization, a VLA-NP sample was negatively stained with 1 wt % uranyl acetate (Electron Microscopy Sciences) on a carbon-coated 400-mesh copper grid (Electron Microscopy Sciences) and visualized using a JEOL 1200 EX II transmission electron microscope. The presence of VLA-4 on WT-NP and VLA-NP was determined using western blotting. First, the samples were adjusted to 1 mg/ml protein content, followed by the addition of NuPAGE 4 lithium dodecyl sulfate sample loading buffer (Novex) and heating at 70C for 10 min. Then, 25 l was loaded into the wells of 12-well Bolt 4 to 12% Bis-Tris gels (Invitrogen) and ran at 165 V for 45 min in MOPS running buffer (Novex). The proteins were transferred for 60 min at a voltage of 10 V onto 0.45-m nitrocellulose membranes (Pierce) in Bolt transfer buffer (Novex). Nonspecific interactions were blocked using 5% milk (Genesee Scientific) in PBS (Thermo Fisher Scientific) with 0.05% Tween 20 (National Scientific). The blots were probed using anti-integrin 4 antibody (B-2, Santa Cruz Biotechnology) or anti-integrin 1 antibody (E-11, Santa Cruz Biotechnology). The secondary staining was done using the corresponding horseradish peroxidaseconjugated antibodies (BioLegend). Membranes with stained samples were developed in a dark room using ECL western blotting substrate (Pierce) and an ImageWorks Mini-Medical/90 Developer. Long-term stability of WT-NP and VLA-NP in 10% sucrose solution was tested by storing the particles at 4C for 2 months with weekly size measurements.

The expression level of VCAM-1 on C1498-WT, C1498-VCAM, untreated bEnd.3 cells, and bEnd.3 cells treated overnight with LPS (1 g/ml) from Escherichia coli K12 (LPS; InvivoGen) was evaluated as described above. For the first binding study, 5 104 cells, either C1498-WT or C1498-VCAM, were collected and resuspended in 160 l of DMEM containing 0.5% USDA FBS, 1% bovine serum albumin (BSA; Sigma-Aldrich), and 1 mM MnCl2 (Sigma-Aldrich). For blocking, anti-mouse CD106 antibody was added to the cells, followed by incubation at 4C for 30 min. Then, 40 l of DiD (1 mg/ml)labeled WT-NP or VLA-NP was added, and the mixture was incubated at 4C for another 30 min. After washing the cells twice with PBS, the fluorescent signals from the cells were detected using flow cytometry. For the second study, 5 104 bEnd.3 cells were plated and then either left untreated or pretreated with LPS overnight. The media were then removed and replaced with 160 l of DMEM containing 0.5% USDA FBS, 0.8% BSA, and 1 mM MnCl2. For blocking, anti-mouse CD106 antibody was added to the cells, followed by incubation at 4C for 30 min. Then, 40 l of DiD (1 mg/ml)labeled WT-NP or VLA-NP was added, and the mixture was incubated at 4C for another 30 min. After washing the cells twice with PBS, the cells were detached by scraping, and the fluorescent signals from the cells were detected using flow cytometry. All data were collected using a Becton Dickinson FACSCanto-II flow cytometer and analyzed using FlowJo software.

Drug loading and encapsulation efficiency were measured using HPLC on an Agilent 1220 Infinity II gradient liquid chromatography system equipped with a C18 analytical column (Brownlee). VLA-DEX-NP samples were dissolved overnight in 80% acetonitrile (ACN; EMD Millipore) and then centrifuged at 21,100g for 8 min to collect the supernatant for analysis. The solutions were run through the column at a flow rate of 0.3 ml/min and DEX was detected at a wavelength of 242 nm. The DEX release profile was obtained by loading 200 l of VLA-DEX-NP (1 mg/ml) into Slide-A-Lyzer MINI dialysis devices (10K molecular weight cutoff; Thermo Fisher Scientific) and floating them on 1 liter of PBS stirred at 150 rpm. At each time point, dialysis cups were retrieved, and their contents were centrifuged at 21,100g for 8 min. The pellets were dissolved in 80% ACN overnight and processed as described above for HPLC analysis.

The biological activity of DEX was evaluated in vitro using a test system involving the LPS treatment of DC2.4 dendritic cells. To validate the system, DC2.4 cells were first plated onto a 24-well tissue culture plate at 5 104 cells per well and cultured overnight with or without LPS at a concentration of 1 g/ml. Then, supernatant was collected, and the concentration of IL-6 was measured using a mouse IL-6 ELISA kit (BioLegend) according to the manufacturers protocol. To compare free DEX and VLA-DEX-NP, the two formulations were first added to the culture medium at final drug concentrations of 0.01, 0.1, and 1 M, followed by 2 hours of incubation. For free DEX, 1000 stock solutions were prepared at 0.01, 0.1, and 1 mM in dimethyl sulfoxide. Then, the cells were treated with LPS overnight before measuring the concentration of IL-6 in the supernatant. To test the effect of empty nanoparticles, either PLGA cores or VLA-NP at a final concentration of 1 g/ml were first incubated with the cells for 2 hours, followed by an overnight incubation either with or without LPS before measuring IL-6 levels.

All animal experiments were performed in accordance with the National Institutes of Health (NIH) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego. To induce lung inflammation in mice, 30 l of LPS (400 g/ml) in PBS was injected intratracheally into male BALB/c mice (Charles River Laboratories). At 1 hour after LPS injection, 100 l of DiD (10 mg/ml)labeled WT-NP or VLA-NP was administered intravenously. After 6 hours, the heart, lungs, liver, spleen, kidneys, and blood were collected. All solid tissues were washed with PBS and suspended in 1 ml of PBS before being homogenized with a Biospec Mini-Beadbeater-16. The homogenates and blood were then diluted 4 with PBS and added to a 96-well plate, and fluorescence was measured using a BioTek Synergy Mx microplate reader. For each sample, the background signal measured from the corresponding organ or blood of control mice that did not receive any treatment was subtracted.

Male BALB/c mice were intravenously injected with 100 l of free DEX or VLA-DEX-NP, each at a drug concentration of 200 g/ml, daily for the first 7 days. Then, for the next 2 days, the dosage was doubled by injecting 200 l of each formulation at the same drug concentration. At 24 hours after the last injection, blood was collected by submandibular puncture and collected into tubes containing sodium heparin (Sigma-Aldrich). Plasma samples were obtained by taking the supernatant of the blood after centrifuging at 800g for 10 min. Creatinine levels were measured using a creatinine colorimetric assay kit (Cayman Chemical Company) according to the manufacturers protocol.

To treat lung inflammation, male BALB/c mice were first intratracheally challenged with 30 l of LPS (400 g/ml) in PBS. At 1 hour after the challenge, 100 l of free DEX, WT-DEX-NP, and VLA-DEX-NP, each at a drug concentration of 200 g/ml, was injected intravenously. After 6 hours, the lungs were collected and homogenized as described above. The homogenates were centrifuged at 10,000g, and the supernatants were filtered through 0.22-m polyvinylidene difluoride syringe filters (CELLTREAT). The concentration of IL-6 was measured using a mouse IL-6 ELISA kit according to the manufacturers protocol. For histology analysis, the lungs were collected after 6 hours and fixed in 10% phosphate-buffered formalin (Fisher Chemical) for 24 hours. The fixed lungs were sectioned, followed by hematoxylin and eosin (Sakura Finetek) staining. Histology slides were prepared by the Moores Cancer Center Tissue Technology Shared Resource (Cancer Center Support Grant P30CA23100). Images were obtained using a Hamamatsu NanoZoomer 2.0-HT slide scanner and analyzed using the NanoZoomer Digital Pathology software.

Acknowledgments: Funding: This work was supported by the National Institutes of Health under award no. R01CA200574 and the Defense Threat Reduction Agency Joint Science and Technology Office for Chemical and Biological Defense under grant no. HDTRA1-18-1-0014. J.H.P. was supported by a National Institutes of Health 5T32CA153915 training grant from the National Cancer Institute. Author contributions: J.H.P., Y.J., R.H.F., and L.Z. conceived and designed the experiments. J.H.P., Y.J., J.Z., H.G., A.M., and J.H. performed all experiments. All authors analyzed and discussed the data. J.H.P., A.M., R.H.F., and L.Z. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper.

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Genetically engineered cell membranecoated nanoparticles for targeted delivery of dexamethasone to inflamed lungs - Science Advances

But I also thought of mosquitoes.

Now I have never been to Florida. But the state is known for its mosquitoes. The humorist Dave Barry lives there and has often mentioned the insects in his columns: ... as the Sun set, we experienced a sensation that I will never forget: The sensation of being landed on by every mosquito in the Western Hemisphere. There were so many of them that they needed Air Traffic Control mosquitoes to give directions."

Long story short: Florida has swarms of mosquitoes. They are constantly biting residents of and visitors to the state, so much so that I feel for the person I know who is going there. Still, get this: in an effort to fight the mosquito menace last April, a biotech firm went to the Keys to release ... more mosquitoes. Hundreds of thousands of mosquitoes, brought to the Keys as eggs actually, allowed to hatch there and live out their lives.

What hare-brained scheme is this, you may wonder. Many people have so wondered, and in the Keys, there has been plenty of oppositionso it is a controversial programme. Yet, it at least deserves some thought, especially given that swarms of mosquitoes are a feature of life in much of India too.

The mosquitoes introduced into the Keys were genetically engineered.

A little background, first. There are plenty of mosquitoes in Florida, certainly, and it cant be pleasant to suffer their bites. But only the species Aedes aegypti actually carries diseaseschikungunya, dengue and moreand they make up only 4% of the mosquito population in Florida. Whats more, only female mosquitoes actually bite humans. Males feed on nectar and their sole purpose in life is to mate with females and produce more mosquitoes. None of this is meant to say that we should ignore these pests. But it does suggest a possible way to fight them thats more efficient than blanket applications of insecticide: target the females.

Its true, the male and the female of the species do look different, but thats if you get a chance to peer closely at them. So, its in no way practical to visually identify only the female mosquitoes in a given area and whack them dead. But what if theres a way to ensure that when a mosquito pair reproduces, the female, and only the female, offspring die? What if such death comes early in their lives, even before they attack humans for the first time? Carnage like this means that the offspring left alive will mostly be males. They will mate with the remaining females, with the same morbid results for the resulting female offspring. Over time, youd expect the mosquito population to become more and more male. With less and less females to mate with, the Aedes aegypti population will naturally decline.

Genetic engineering (or genetic modification) offers a way to accomplish more or less this. Though with various plant species especially, plenty of controversy surrounds the process. Consider:

Proponents point out that humans have been doing such engineering indirectly for many millennia: breeding plants and animals selectively for certain desirable characteristics. For example, modern corn looks nothing like the grass-like Mexican plant with rudimentary ears, teosinte, that it is descended from. Thats because we humans have for uncounted generations selected plants with juicier, bigger and more succulent ears and kernels and used only those plants to generate their next crop. Much the same applies to plenty of other crops and domesticated animals.

Critics, though, say that todays techniques of actually modifying genes are entirely different from selective breeding, and theres definite danger there. For example, the wind can carry pollen from genetically modified (GM) crops to fields of non-modified crops, causing unpredictable and undesirable problems. Besides, the GM crop industry is dominated by a few large biotech firms. So, the prospect of widespread use of such crops raises serious concerns about monopolies, especially for small farmers like in India.

The fear that genetic engineering can have unpredictable consequences is why many residents of the Keys opposed the new genetically-engineered male mosquitoes.

Still, lets look at how they were engineered and then released. These Aedes aegypti males have had their DNA altered: scientists have edited" two particular genes into particular locations in the mosquitos genome:

* a fluorescent marker" gene that glows in red light, which will later be used to identify engineered mosquitoes.

* a self-limiting" gene.

When the insects reproduce, both genes are passed on to their offspring. The self-limiting" gene has no effect on males. But in larval females, it inhibits the storage of a specific protein that would otherwise build up as the insect grows. The result is that the female dies before it can mature.

This is the theory, of course. But these engineered mosquitoes have been released in Brazil, Panama and even Indiain the last two years, over a billion of them. The British biotech company that produced them, Oxitec, reports that in those areas, the populations of Aedes aegypti shrank by over 90%. Youd think that would certainly have an effect on the incidence of mosquito-borne diseases.

What of unpredictable consequences? The Brazil trial suggested that the self-limiting gene did not kill all the female offspring before they could mate, because other genes from the engineered mosquitoes appeared among other local mosquitoes. What effect this will have on the local ecosystem is not yet clear. But this is the kind of fallout of genetic engineering that worries many people.

Still, in April, Oxitec placed boxes containing eggs of the engineered mosquitoes in six different locations in the Florida Keys. Each week between May and August, about 12,000 of the mosquitoes will hatch from their eggs and emerge into the Florida air, ready to find willing females to mate with. Every now and then, Oxitecs researchers will collect mosquitoes and use red light to identify the engineered ones. They want to know such details as their life spans, the distance they have travelled from their boxes, and how many of the females who inherit the self-limiting gene have actually died. All this will shape a second and larger trial later this year, when Oxitec plans to release 20 million engineered mosquitoes. Data from these trials will help decide whether it is worth releasing mosquitoes more widely across the US.

Clearly, theres still plenty to learn about genetically engineered mosquitoes. But till now, insecticides have been our weapons of choice against mosquitoes. They kill the insects, sure, but also other insects we would rather save, like honeybees.

Consider this parallel to cancer. Our weapon of choice there one thats just as blunt as insecticidesremains chemotherapy. It kills cancer cells, sure, but also plenty of other cells in our bodies. What if we instead found a way to introduce a particular kind of cancer cell into the body, one that would single out and kill the malignant cells?

We dont know of such a cell (yet, anyway), but thats how to think of genetically engineered mosquitoes. And if you think about it some more, theres also a parallel of sorts to vaccines for a certain virus that we are all a little too familiar with these days.

Once a computer scientist, Dilip DSouza now lives in Mumbai and writes for his dinners. His Twitter handle is @DeathEndsFun

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Get the females and beat the disease - Mint

Every year, billions of pounds worth of potatoes are chucked in the bin because of late blight disease. The disease, which rots potatoes from the inside out, was one of the major causes of the Irish potato famine and is still a scourge on spuds today. But, in fields in Norwich and Cambridgeshire, experimental blight-resistant potatoes are growing unbothered by the disease. The only problem? No one can eat them.

These potatoes were created by taking genetic material from one organism and inserting it into another one in this case blight-resistance genes were whisked out of a wild potato relative and put into Maris Pipers. Under EU and UK law, these kinds of crops are defined as genetically-modified organisms (GMOs) and are subject to strict regulations that limit how they are grown and whether they can be sold as food.

Boris Johnson has these potatoes or at least crops like them in his sights for a while. On the very first day of his premiership, Johnson signalled that his government would like to break away from EU laws on GMOs. Let's start now to liberate the UK's extraordinary bioscience sector from anti-genetic modification rules, and let's develop the blight-resistant crops that will feed the world, Johnson said in his first speech as prime minister. On June 17 the government is poised to announce its response to a public consultation on genetic engineering the first step in what might turn out to be a major re-working of the UKs genetic engineering laws.

But heres where things get tricky. While the changes proposed by the Department for Environment, Food & Rural Affairs (Defra) would change the way that genetically-engineered crops are defined in the UK, theyre unlikely to clear a path for blight-resistant potatoes to come to our shelves any time soon. The government is keen to show that its blowing away the cobwebs of EU legislation, but in reality the trickiest scientific and legal questions are all still to come.

At the heart of the consultation is a question about how to regulate crops and animals that have had their genomes changed using precision gene-editing techniques such as Crispr. At the moment, EU (and UK) law makes no distinction between Crispr-edited crops and those made using older forms of genetic engineering such as transgenesis the same technique used to make those blight-resistant potatoes. Under EU law, crops made using either of these techniques are currently classified as genetically modified organisms (GMOs) but Defra is proposing that some gene-edited crops should not be classified as GMOs.

This change in regulations would be a relief for UK researchers who work on genetically-engineered plants, says Wendy Harwood, leader of the Crop Transformation Group at the John Innes Centre in Norwich. Under current rules, researchers wishing to plant GM crops in field trials must go through a lengthy approval process and then follow strict rules, including leaving test fields empty for a year after the crop has been harvested. Although the EU does allow the growing and marketing of GM foods, the approval process is so long and expensive that many companies effectively consider the EU a closed market to GMOs.

As a result, field trials of genetically-engineered crops are rare in the UK. As of November 2019, only three UK-based research departments had consent to hold field trials involving GMOs. Knowing those chances might have increased a bit, it might be a bit easier just to take that next step, says Harwood.

A change in the definition of GMOs would bring the UK more in line with countries like the USA, which allows Crispr-edited crops to sidestep regulation governing GMOs. Jonathan Jones is a professor at The Sainsbury Laboratory in Norwich and leads the team behind the late blight-resistant potatoes. He says that genetically-engineered crops can play a major role in reducing agricultures impact on the planet. The real problem comes from doing agriculture. Actually, what we want is less agriculture. And the way to have less agricultural and more land set aside for biodiversity is to have our agriculture more productive, he says.

The rest is here:
The UKs big GMO food plan might not be that big - Wired.co.uk

New research from a University of Minnesota team shows T cells can be engineered to migrate more effectively inside tumor microenvironments, raising hopes of reproducing the remarkable outcomes seen for certain hematological cancer patient subsets in broader populations and indications.

T cells have been key to two promising families of cancer immunotherapies, chimeric antigen receptor (CAR) T cells and immune checkpoint inhibitors. After two new U.S. Food and Drug Administration (FDA) approvals earlier this year for Bristol Myers Squibbs Breyanzi (lisocabtagene maraleucel) and Abecma (idecabtagene vicleucel), there are now five marketed CAR T cell therapies for a range of hematological cancers, including large B-cell and mantle cell lymphoma, acute lymphoblastic leukemia and multiple myeloma. These therapies are patient-derived T cells engineered to express a CAR that directs them to a specific tumor.

By contrast, immune checkpoint inhibitors function by targeting sets of natural immune system brakes, either on a tumor or directly on immune cells. The seven FDA-approved immune checkpoint inhibitors allow T cells to kill a growing list of hematological and solid tumors, and show impressive outcomes but in a fraction of patients.

Both approaches have been more effective in hematological cancers, and one obstacle in solid tumors is a literal barrier. T cells have to migrate through the complex, dense, and rigid tumor microenvironment, which can misdirect and slow down the immune cells.

The new paper, published in Nature Communications, shines a light on how T cells move within these physical features, informing new methods to rationally engineer the cells for more effective therapies.

T cell engineering has focused on cancer identification and targeting, said Paolo Provenzano, associate professor at the University of Minnesota Masonic Cancer Center and an author on the paper.

Were trying to add on a piece to that: they know what to kill, how do they get there? he said.

The researchers looked at pancreatic ductal adenocarcinoma (PDAC), a particularly fibrotic cancer known for limited cytotoxic T cell infiltration. Provenzano and colleagues had previously shown that antifibrotic therapies could improve immune cell infiltration in PDAC, but the group is now focused on rationally designing physical attributes of the T cells themselves.

Provenzanos team started by testing T cell migration on specialized artificial 2-D nanotextured platform that mimicked elements of the stromal extracellular matrix of tumors. T cells use lamellipodia and filopodia to sense biophysical cues and guide migration, and can switch between more flexible and stiffer phenotypes, to adapt to different conditions.

Theyre never all or one, theyre on this sliding scale, mediated by the muscle-like contractions of microtubules, Provenzano said.

The researchers found that while in a more amoebalike phenotype, cells had enhanced speed and motility, improving how quickly and effectively the cells could navigate the matrix. The researchers used the chemotherapeutic nocodazole, known to destabilize microtubules, to increase contractility and induce more amoebalike phenotypes, and another chemotherapeutic, paclitaxel, for the inverse. Cells with the nocodazole-induced amoebalike phenotypes could contract into smaller balls, and could shoot out protrusions faster, allowing for better motility than the paclitaxel-induced stiffer ones, and migrating between 50-100% faster in artificial 3-D matrices and mouse PDAC model tumor slices.

Thinking about personalized combination approaches to cancer treatment, this raised a red flag for Provenzano, as taxane agents are commonly used to treat solid tumors like breast and pancreatic cancer. The findings suggest certain chemotherapies might not be right if youre trying to elicit the bodys own immune response against these tumors, or mixing with a T cell therapy, he said.

The effect of chemotherapies on T cell microtubules is transient, so next the researchers explored a genetic engineering approach, using CRISPR technology to engineer T cells lacking GEF-H1, a gene they hypothesized mediates cell contractility. In a 3-D matrix, the knockout lines migrated 50% compared with controls.

Provenzano envisions different T cell engineering tweaks as part of the increasingly personalized approach to cancer treatment. Cells engineered to migrate faster have promise particularly in fibrotic tumor microenvironments, where T cells move about half as fast.

They get to sample less volume before they become exhausted, he said.

T cell exhaustion is a key limitation for CAR-T therapies and combining them with T cells that are physically optimized for faster migration could be synergistic in these patients.

If tumors are like unique obstacle courses for T cells, though, then different athletes or teams might be suited for different cancer types and patients.

Maybe we engineer five different T cells that navigate different obstacles, then mix them in cocktail and bring them back in. Thats what were thinking as our ultimate goal, he said.

Provenzanos lab is now using physics-based mathematical models to predict what the optimal cell would be.

We dont know the most physically optimized cell looks like yet, he said. But the paper represents just the first foray into the optimization process for just the first athlete. This will be what at least half my lab will be publishing on for the next decade.

There is active interest in the approach, but Provenzano said the work is not yet being commercialized. He hopes to find a partner with an ongoing T cell therapy clinical trial that could add an arm with his groups physically optimized cells, but trials are probably still years away.

Link:
Tumors Next Target for T Cell Therapies with U Minnesota Research - BioSpace

The University of SydneysCharles Perkins Centrewas named the recipient of theinauguralNHMRC Research Quality Award. The biennial award recognises excellence and outstanding contribution to ensuring the highest quality in health and medical research.

Selected by the NHMRC CEO in consultation with the Chairs of NHMRCs Research Committee and Research Quality Steering Committee, the award acknowledges the significant contribution the Centre has made to actively pursuing a research culture conducive to the highest quality research involving the use of animals.

To support its commitment to conducting high-quality animal research, the Charles Perkins Centre commenced a research and development program to implement actions recommended by NHMRCs best practice methodology in the use of animals for scientific purposes. Led by Associate ProfessorKieron Rooney, and working with local and international collaborators, the team is investigating the barriers and facilitators to engagement with pre-clinical trial registration and developing educational tools.

The program is delivering a cultural shift in the conduct of animal research that will enhance transparency and reproducibility, reduce animal use and, ultimately, deliver improvements in human health.

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Sydney researchers receive prestigious NHMRC awards - News - The University of Sydney

Capsida CEO,Dr. Robert (Bob)Cuddihy/ Photo Courtesy of Capsida Biotherapeutics

Capsida Biotherapeutics, which debuted in April already holding a research alliance with AbbVie, has wasted no time in putting its fully integrated adeno-associated virus (AAV) platform to work in the highest need therapeutic spaces.

This morning, Capsida announced a strategic collaboration with gene therapy leader, CRISPR Therapeutics to develop, manufacture and commercialize in vivo gene-edited therapies for amyotrophic lateral sclerosis (ALS) and Friedreichs ataxia (FA).

Per the agreement, CRISPR will lead the research and development of the Friedreichs ataxia program, while Capsida will shepherd the R&D for the ALS effort. Capsida, of course, will conduct the capsid engineering for both programs and will also assume responsibility for the process development and clinical manufacturing of both programs.

These are two neurodegenerative targets with significant unmet needs, and Capsida Chief Executive Officer, Dr. Robert M. Cuddihy, is excited about the progress this collaboration could bring.

I think it's going to take a different and a novel approach, and I think we're combining two innovative approaches that hopefully can really be disruptive to the field and start to bring some benefit to patients, he said.

Maybe the lesser-known of the two indications, Friedreichs ataxia is a genetic, progressive neurodegenerative movement disorder caused by an expansion of a GAA trinucleotide repeat in the FXN gene. This bears similarity to a familial form of ALS that the partners will be looking at.

What we can do in both of those diseases with genome editing is essentially excise the pathogenic repeat out of the genome to return the genome to more or less its non-aberrant state, said CRISPR Chief Operating Officer, Dr. Lawrence Klein.

CRISPR has done extensive work on the gene-editing side of bothdiseases. The next challenge, Klein shared, is to deliver the machinery necessary for editing, and Capsida stood out as having these particular capabilities.

We looked at a number of different companies that are working on engineering AAV to be more neurotropic, and the data that Capsida has been able to generate through their engineering platform really stood out. The neurotropic capacity of their engineered AAV vectors is truly remarkable, he said.

Capsidas high-throughput engineering and cargo development platform is designed to improve the safety, specificity, and reach of gene therapies for currently inaccessible targets.

By selecting the delivery vehicle, a novel engineered capsid, that has significantly enhanced tissue specificity versus AAV9, while de-targeting the tissues not relevant to the targeted disease, we expect potentially enhanced efficacy and safety profiles, Cuddihy explained.

In two diseases with no approved disease-modifying treatment options, time is of the essence, and Capsida, which has its own GMP manufacturing facility, could help to expedite time-to-market for a successful therapy down the line.

We pride ourselves on really being a fully integrated gene therapy company. Not only are we selecting novel capsids, we're assessing them upfront for manufacturability, ability to scale. We have our GMP facility opening up this year, so we're raring to go, Cuddihy said.

In this evenly-yoked partnership, both CRISPR and Capsida will have the option to co-develop and co-commercialize each program. Following such an option, the two would equally share all research, development, and commercialization costs and profits of an approved product.

I think there is a high degree of unmet need where small molecules and antibodies haven't had as much success, and where gene therapy and genome engineering can really bring about new approaches to treat these diseases, Klein said. Delivery technologies are getting better and better where I think we can really start to access the targets in a way that doesnt bring toxicity challenges.

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CRISPR and Capsida Partner in Gene Edited Therapies for ALS, Friedreich's Ataxia - BioSpace