Daily Archives: June 18, 2021

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

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

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.

Read this article:
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.

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

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The Center for Food Safety is the kind of organization that most progressive foodies can get behind: Its website features photos of graceful monarch butterflies and dairy cows with big, doleful eyes. Its recent campaign slogans implore supporters to tell EPA to stop this brain-damaging pesticide! and protect dolphins and birds from floating factory farms! It advocates for farmworker rights, humane treatment of animals, and protection of pollinators.

Oh, yes, and theres one more thing: The 24-person nonprofit, whose revenue in 2019 was about $5.2 million, wants the US government to stop supporting certain kinds of high-level virology research. Last month, the group sued the National Institutes of Health in an attempt to force the agency to reveal information about its funding for what is known as gain-of-function researchthe term refers to a category of lab work that seeks to understand how viruses create pandemics. Sometimes, but not always, the research involves manipulating viruses to make them more virulent and contagious to study how they evolve.

Virologists say this kind of research is vital and has led to many important medical discoveries, including during the COVID-19 pandemic. But Center for Food Safety argues that gain-of-function research is too dangerous to pursue. A pathogen released from a lab could result in catastrophic consequences to the human environment, CFS staff attorney Victoria Yundt warned in a recent press release about the NIH law suit. Andrew Kimbrell, a public interest attorney who founded the Center for Food Safety in 1997, was also quoted in the release saying, The NIHs refusal to make public the research it is funding to enhance the transmissibility, infectiousness, and lethality of potential pandemic viruses is grossly irresponsible.

Why would this lefty food and farms groupand they arent alonerail against high-level virology research? The key to the answer has to do with the Center for Food Safetys long opposition to the practice of genetic engineering. In a recent phone call, I spoke to CFSs Kimbrell, who explained what he sees as the connection. You genetically engineer bacteria and plants, then you genetically engineer animals, then you genetically engineer embryosall that has happened, with some promise, but also a tremendous amount of danger and threat, he said. Now, viruses are not technically an organism, but they are living biological elements. So, they fit certainly within that narrative: Just because we can do something doesnt mean we should do something.

Kimbrell said he absolutely thinks the pandemic was the result of an accidental lab release. Scientists at the Wuhan Institute of Virology, he believes, used gain-of-function to enhance a coronavirus. The virus then escaped out of the lab, spread uncontrollably, and caused the COVID-19 pandemic. Hence, in effect, gain-of-function research caused the pandemic.

Kimbrell isnt the only genetic-engineering critic who has come out against gain-of-function research and in favor of the lab-leak theory. Over the past few months, many organizations that once busied themselves warning the public about pesticides in breakfast cereal and the evil deeds of Monsanto have pivoted to protesting gain-of-function work. Organic Consumers Association, a nonprofit that advocates against pesticides and genetic engineering of food, published a hall of shame series on virology researchers who use the technique. As we follow the evidenceand follow the moneywe come face to face with a cast of out-of-control Mad Scientists, militarists and biotech/bio-pharmaceutical entrepreneurs, one recent post reads.

US Right to Know, a nonprofit that advocates for transparency around genetically modified food, announced in May that it is expanding its investigative work into other urgent public health matters, including the origins of the novel coronavirus SARS-CoV-2, which causes the disease COVID-19. Robert F. Kennedy, Jr.s anti-vaccination advocacy group Childrens Health Defense, which has also criticized genetic engineering, has taken up the campaign against gain-of-function and embraced the hypothesis that these experiments made COVID-19 more dangerousnot to mention that it then may have escaped from a lab. The website of the International Center for Technology Assessment, an anti-GMO group that Andrew Kimbrell founded alongside Center for Food Safety, now seems almost entirely devoted to criticizing gain-of-function and promoting the lab-leak hypothesis.

Over the last year, I have watched online anti-vaccine groups radicalize parents, starting with questions about shots and then moving them into pandemic-denying conspiracy theory adherents. Ive noticed that even in our politically polarized world, the political spectrum isnt always a straight line; sometimes its a circle where at a certain point in the back, the far left and the far right converge.

I am now watching the same dynamic play out in anti-GMO groups, as environmentalists who worry about pollinators get drawn into a dark narrative that vilifies vital research. With tens of thousands of followers on social media, anti-GMO groups have the potential to turn the tide of public opinion; hanging in the balance is science that could potentially help prevent the next pandemic.

Also at stake is the reputation of scientists themselves. When I asked Kimbrell why researchers would willingly undertake gain-of-function experiments if they really were both dangerous and unnecessary, he told me that he believes that the basic motivation could be found in the inflated egos of researchers. For some reason, theres always this altruistic halo around scientists, but you know, they created nuclear weapons, science has created all the chemicals that are polluting our air and our environment, he said. I think theres a huge amount of sort of just, Lets do it, because we can and its exciting. Like crossword puzzles.

Since the lab-leakhypothesis has dominated the news recently, let me back up. My suspicion is that no one will ever know for sure where the virus came from, though most scientists agree that the United States and China must conduct a thorough investigation into its origins. Scientists also agree about the paramount importance of lab safety; if youre interested in learning more about lab leaks, I urge you to read this piece by Rowan Jacobsen that Mother Jones published a year ago.

To be fair to the opponents of gain-of-function research, most experts agree that some extreme versions of this work are truly reckless. As Jacobsen wrote, lab leaks have occurred with some frequency over the past few decades. An early controversy over gain-of-functionresearchcame to a head in 2011, when University of Wisconsin researcher Yoshihiro Kawaoka announced that his lab had successfully modified the highly lethal H5N1 bird flu to make it airborne among ferrets. After much media criticism of this experiment, in 2014, the Obama administration placed a moratorium on most gain-of-function experiments the US funded throughout the world. The Trump administration mostly lifted the restriction in 2017, though at the onset of the pandemic, the former president decried the practice and claimed that the US funding of such work in China was a relic of the Obama administration. (That wasnt exactly true; after all it was his administration who did away with the moratorium.)

Last month, after more stories about the Wuhan lab appeared, the US Senate approved an amendment put forth by Sen. Rand Paul (R-Ky.) that would permanently halt US funding of gain-of-function research in China. While many still deny funding gain-of-function research in Wuhan, experts believe otherwise, Sen. Paul said in a press release. The passage of my amendment ensures that this never happens in the future.

Yet the virus researchers I spoke with emphasized that not all gain-of-function research is dangerousto the contrary, most of it is quite routine and has been crucial in advancing disease research and treatment. Gerald Keusch, an infectious disease specialist who is a co-director of Boston Universitys National Emerging Infectious Diseases Laboratories, points out that the term gain-of-function is frustratingly broad and describes a large swath of virus work. Most scientists will say that gain-of-function is too crude a terminology, he told me. It encompasses things that are necessary and safe, to things that are necessary but somewhat risky, to things that are maybe not necessary and highly risky. And they all get wrapped under the same term. He points out that some landmark medical breakthroughs happened because of gain-of-function: For example, the technique led to University of North Carolina scientists discovery that the drug remdesivir could treat COVID-19. Indeed, work under the broad category of gain-of-function also may have contributed to the vaccine research on other coronaviruses that hastened the development of the COVID-19 vaccines, Keusch added.

The outcry over gain-of-function research has cast a pall of suspicion over other, completely unrelated kinds of pandemic research. Peter Daszak, thepresident of nonprofit EcoHealth Alliance, has seen this play out firsthand. EcoHealth Alliance, which has an annual budget that ranges from $10 million to $17 million, a US-based staff of about 50, and some contractors abroad, came under scrutiny last April, when media reports revealed that the organization had earmarked about $600,000 of a $3.4 million NIH grant to fund preapproved research at the Wuhan Institute of Virology. In the wake of these reports, the Trump administration canceled the entire NIH grant for EcoHealth Allianceeven the parts that had nothing to do with the Wuhan lab.

This move struck Daszak as bizarre. We dont even have a lab, he told me. We do things like produce a book on how to live safely with wildlife or educate community leaders. Despite the actual nature of his work, over the past year, he and EcoHealth Alliance somehow have become the symbols of gain-of-function research. The GMO-transparency group US Right to Know recently filed 74 requests under the Freedom of Information Act that tied up EcoHealths board members and funding agencies for months. They know that a small organization like ours cannot keep up with that level of work, Daszak said. He finds irony in anti-GMO groups crusade against his work. Youve got these anti-GMO groups trying to shut us down when here we are trying to achieve the same goal, he said, which is a more balanced relationship with nature.

The overlap that Daszak describes reflects a complicated political dynamic. The anti-GMO movement used to reside comfortably on the left: A 2001 ABC News survey found that Democrats were far more likely than Republicans to believe that genetically modified foods were unsafe, and Democratic politicians have fought hard for the mandatory labeling of these foods. Science writers have called the movement against GMOs the lefts anti-science problem. But that may be changing: A 2016 Pew survey found that equal numbers of Democrats and Republicans believe that GMOs are worse for peoples health. That same year, when the Senate was divided over a controversial GMO-labeling bill, many Democrats argued it didnt go far enough in requiring companies to disclose genetically engineered ingredients. But others defended the measure. It will provide fair and objective information without stigmatizing foods that are completely safe, Indianas Democrat Sen. Joe Donnelly said. Sen. Debbie Stabenow (D-Mich.) spoke of the scientific consensus that biotechnology is safe. (The bill passed 6330, and the law set to go into effect later this year.)

But now, this past year, anti-GMO groups gain-of-function criticism has drawn more attention from the right. Center for Food Safetys Kimbrell said that in the weeks since his group sued the NIH over gain-of-function transparency, he has been invited to speak on several right-wing talk shows. (He declined.) And until recently, the idea that COVID-19 could have come from a lab was considered mostly a right-wing theory, promoted with a few racist dog whistles by President Trump and his supporters. Anti-GMO groups dont typically endorse that kind of rhetoric, but with their embrace of the lab-escape theory, they couldnt help but attract it. I noted a xenophobic flavor to many of the comments on several of the groups social media posts referencing COVID. In response to a recent US Right to Know post, one commenter referred to COVID-19 as the China virus and President Biden as China Joe.

Peter Hotez, a vaccine scientist and misinformation expert with Baylor College of Medicine points out that the uproar over gain-of-function research plays right into the conservative narrative about greedy pharma companies and power-hungry scientists. Hotez notes that the right-leaning anti-vaccine groups that he tracks have begun to embrace anti-GMO rhetoric, insisting that mRNA vaccines genetically modify humans. (They dont.) In the past we said, this is far left and far right, but I am seeing more far-right dominance. Its becoming much more of a mainstream GOP dynamic.

Kimbrell doesnt see it this way. His group has always attracted a small contingent of libertarians, he said, accounting for perhaps 5 or 10 percent of the supporters. He noted that he has encouraged his staff to get vaccinated against COVID-19; he opted for the Johnson & Johnson shot because he was skeptical of the mRNA technique the Moderna and Pfizer used to develop their shots. Were bipartisan, were trying to have humans, as a species, live in a mutually enhancing world with the rest of nature, and thats generally viewed as progressive, he said. Im not quite sure why.

Progressives have a long history of advocating for diplomacy and cooperation over threats and hostile overtures. But these are strange times, with the far left and the far right bonding over vaccines, and conspiracy theories, and now, pandemic virology research. This dynamic can be dangerous not only to science, but also to the researchers who conduct this work.

In recent months, the paranoia about EcoHealth has spilled over into Daszaks personal life. He and his staff have received dozens of death threats. After someone sent an envelope of white powder to his home, he decided to hire security personnel to protect his family.

Baylors Hotez noted that threats to scientists have increased dramatically over the past year. He says that when prominent activists cast scientists as evil power brokers, it fosters an atmosphere of animosity toward researchers. Anti-science groups are trying to paint a picture of scientists as evildoers rather than the humanitarians that most of us are, he said. Most of us became scientists to help, to do something for humanity. Hotez himself has received many recent threats related to his vaccine advocacy, some of them anti-Semitic in nature.

Beyond his own personal safety, Daszak worries about the implications for infectious disease research: If the United States stops virology work in China its likelyscientists accessto a place where these viruses come fromwill be blocked. The tensions over the lab-leak theory may escalate to the point where scientific cooperation breaks down. And well certainly never get to conduct a thorough investigation into the origins of the pandemic, Boston Universitys Keuschsaid,noting, The best hope is that somehow in the back channels, we will figure out how to lower the heat, increase the light, convince the Chinese its in their interest to be transparent.

Will the diplomatic back channels prevail in time to prevent the next pandemic? Thats anybodys guess, because pathogens emerge, change, and spread all around us, all the time. Just last week, Chinese authorities reported the first human case of a rare bird flu. The idea that the United States wouldnt be able to monitor and study this pathogen alarms Daszak. If we dont do that work, its really damaging to future public health risk, he said. Well never know whats coming next.

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Inside the Anti-GMO Movement's Obsession With Virology Research and Lab Leaks Mother Jones - Mother Jones

DUBLIN--(BUSINESS WIRE)--The "Global Biohacking Market (2021-2026) by Product, Application, Type, End-User, Geography, Competitive Analysis and the Impact of Covid-19 with Ansoff Analysis" report has been added to ResearchAndMarkets.com's offering.

Key factors, such as the innovative trends of neuro-nutrition & growing biohacking in the health & wellness space, are likely to contribute to the growth of the market. Penetration of the Internet of Things in healthcare, fitness, and consumer electronics such as fitness bands also boosts the market growth. Rising demand for smart devices and effective drugs to meet the daily healthcare needs amongst the population and the prevalence of chronic disorders are factors driving the market growth. The COVID-19 pandemic has led to a surge in market growth for the pharma industries engaged in biohacking.

However, strict government regulations governing the genetic engineering experiments, lack of funds required for research, and lack of expertise are likely to hinder the market growth. Cybersecurity practices are expected to pose a challenge for the biohacking market.

Market Segmentation

The Global Biohacking Market is segmented further based on Product, Application, Type, End-User, and Geography.

Why buy this report?

Market Dynamics

Drivers

Restraints

Opportunities

Challenges

Companies Mentioned

For more information about this report visit https://www.researchandmarkets.com/r/ked5i

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Global Biohacking Market (2021 to 2026) - Lab Experiments with The Use of Medical, Nutritional, and Electronic Technique Present Opportunities -...

Pixabay // Qimono

Admit it at some point in our childhoods, weve all wondered if we had a long-lost twin or a celebrity relative. Perhaps weve gone so far as to perform a genetic test through a company such as 23AndMe, and learned more about our ancestry and distant relatives. Though we often pursue genetic testing for personal purposes, finding out individuals that share DNA could have grand implications for our understanding of the health and structure of human populations at large.

This process of examining genetic connections has been streamlined by the introduction of iLASH (IBD by LocAlity-Sensitive Hashing), an algorithm that efficiently identifies relevant genetic connections among large sets of people, which then inform important advancements in population genetics and personalized medicine, among other fields.

What is iLASH?

iLASH came to be when Ruhollah Shemirani, Ph.D. student at USC, teamed up with Jose-Luis Ambite, Research Team Leader at USCs Information Sciences Institute (ISI) and Associate Research Professor of Computer Science at USC Viterbi, to study how genetic connections among individuals can shed light on genetic causes of diseases and the genetic structure of populations. In addition, iLASH can also be used for other purposes such as finding distant relatives through services like 23AndMe. This project was made possible by collaborating with experts from the University of Colorado, the Icahn School of Medicine at Mount Sinai, and the University of North Carolina at Chapel Hill.

Establishing genetic connections can make the world feel smaller than you think. Ambites experience, among those of many others who have used similar services, serve as interesting examples.

According to 23andMe, I share 0.07% of my genome with Greg Ver Steeg, another researcher at ISI, mentioned Ambite. Greg is American (from Dutch descent, many generations ago). Im from Spain. Nonetheless, we share a bit of DNA from a common ancestor.

Essentially, iLASH is a method for IBD, or Identity-By-Descent, estimation. IBD estimation is the process of finding out where, and how much, each pair of individuals in a genetic dataset share their DNA due to shared ancestry, explained Shemirani.

IBD estimation is the first step of IBD Mapping, a novel process to identify the genetic basis of disease. This process is broken down into three steps, each to be published independently in a paper. The first step, published as a featured article in the Editors Highlights in Nature Communications on June 10th, involves estimating genetic segments that are shared between pairs of individuals using iLASH. Next, this genetic pairing information is used to create groups of distant families using network clustering methods. The last paper will focus on statistical methods to show whether these distant families reveal elevated rates of diseases or other traits.

A Pioneer in IBD Estimation

So what sets iLASH apart from other genetic algorithms? Scalability and accuracy.With the ability to perform IBD estimation on a large-scale, biobanks, or storages of biological samples for research, that were previously unfeasible can now be analyzed for genetic connections at an unprecedented speed.

Before iLASH, finding genetic connections in a dataset of 50,000 individuals would take more than a week (~6 days per chromosome), said Shemirani. The same dataset is analyzed by iLASH in an hour!

To achieve this, iLASH employs Locality Sensitive Hashing (LSH), which eliminates unrelated pairs of genetic samples, leaving remaining pairs that have a high probability of shared DNA. This complex algorithm has been facilitated by parallel computing, which allows multiple processes to be carried out simultaneously, creating an efficient approach to IBD estimation.

As a crucial step, Shemirani and Ambite collaborated with geneticists and researchers from various institutions to ensure iLASH is compatible with common formats used by bioinformaticians, who apply information generated from the algorithm to biological and medical research.

Without such feedback from real geneticists at the University of Colorado Medical Campus and Icahn School of Medicine at Mount Sinai, we could not have achieved this, Shemirani said.

Revolutionizing Population Genetics

iLASH has significant real-world applications in both population genetics and personalized medicine.

In the population genetics field, iLASHs efficiency and accuracy as an IBD estimation method has been unprecedented by other types of analyses and has already been implemented by experts across the country.

We can use iLASH in very large datasets to extract patterns of migration and recent fine-scale ancestry structures for the first time, said Shemirani.

In fact, Dr. Gillian Belbin, an iLASH co-author and researcher at the Icahn School of Medicine at Mount Sinai, used iLASH to analyze the UK Biobank, a genetic dataset of 500,000 people in the UK. Among other findings, the study showed patterns of common ancestry with Nordic populations who inhabited areas that are historically contact points for Viking populations.

Incorporating Diversity into the Conversation

In the field of medicine, iLASH is not only an effective tool for studying the genetic origins of rare diseases, but also a promising way to better our understanding of diversity in genetics.

Helping with the discovery of these rare genetic origins for various diseases is just one of the utilities of such studies, Ambite noted. For example, they can also help geneticists with calibrating genetic disease risk calculations for diverse non-European populations.

By building upon previous analyses that were limited to white European populations, iLASH enables researchers to expand existing results to cover a broader range of population groups.

Including iLASH in genetic study pipelines, such as polygenic risk scores or disease mapping studies, will help to account for population structure and hidden relatedness in the datasets, explained Shemirani. This will help partially address the problems that arise from the imbalance, or lack of diversity, of the datasets and studies in terms of population demographics.

Another upside of iLASH is that its more cost-friendly when compared to many other alternatives in medicine, therefore making it a much more accessible option.

Going Forward

Though iLASH has proven to be highly promising in various applications, there is still work to be done. Shemirani named three particular improvements that they are currently working on.

The primary challenge is to create a distributed version of iLASH to meet increasing scalability demands. As datasets grow larger by the day, iLASH needs the resources required to cover a sizable amount of data accurately and efficiently.

In addition, Shemirani and Ambite are also looking to create a cloud service for iLASH, though ethical and security issues surrounding sensitive genetic data pose a problem for this goal.

Finally, adding an incremental analysis would allow iLASH to be adopted in commercial settings where new customers are constantly being added and need to be incorporated into the existing dataset.

Though not all of us are going to find a lost-long twin or celebrity relative, iLASH can help researchers extract crucial genetic information that will inform relevant research in the fields of population genetics and medicine, benefiting us all in the long term.

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Find Your Genetic Relatives Quickly and Accurately with USC ISI's iLASH Algorithm - USC Viterbi | School of Engineering - USC Viterbi School of...

With the rising acreage of hybrid tolerant Bt cotton (HT Bt cotton) threatening to cut into the revenues of seed firms and yields for farmers, seed industry associations have appealed to the Union government to take measures to curb HT Bt cultivation.

The National Seed Association of India (NSAI) and the Federation of Seed Industry of India (FSII), have alleged that the sale of HT Bt cotton seeds has suddenly shot up this year, posing a serious threat to the environment, farmers, legitimate seed companies and government revenue. It cautioned that consequences of allowing the illegal technology could be disastrous.

Also read: Highest HTBT cotton sowing in coming kharif: Shetkari Sanghatana

The two associations have written a letter to the Union Ministry of Agriculture, the government of India and the GEAC (Genetic Engineering Approval Committee) to initiate steps to curb the sale of the illegal seeds and take action against the offenders.

Besides causing harm to the environment and losses to the industry, the HT Bt seeds could contaminate the production of legitimate seeds.

The sale of HT Bt cotton is illegal in the country as the GEAC has not given permission to the technology yet.

The HT Bt cotton, the third generation biotechnology in cotton, gives the plant an ability to survive specific herbicides. It works on the premise that when a specific herbicide is sprayed on the crop variety/hybrid modified to resist that herbicide survives and all others are killed. The genetically modified plant has strength to withstand the effect of weedicide.

Though the farmers in some States have been growing HT Bt cotton for the last few years, the extent was limited to some pockets.

The industry associations alleged that there is a sudden surge in the acreage. Quoting a Department of Biotechnology (DBT) survey, they said about 15 per cent of the cotton acreage in Maharashtra, Andhra Pradesh, Telangana and Gujarat was under HT Bt cotton.

The area under cultivation of illegal HT cotton has been increasing over the years. However, this year there is a big jump in such illegal cultivation especially in the major cotton States, M Ramasami, Chairman of FSII, said.

From an estimated 35 lakh packets last year, the area is likely to go up to about 70 lakh packets this year, Ramasami, who is also the Chairman of the Rasi Seeds, said.

The fact that the total size of the cottonseed packets is five crore packets (of 450 gm each) shows how alarming the situation is.

He said that the illegal seed packets indicate the presence of several technologies in the seeds. This could pose a very serious challenges to the environment.

If it is not controlled immediately by the government, it will spell disaster for the industry and farmers, he said.

NSAI President Prabhakar Rao cautioned that the proliferation of illegal seeds could decimate small cotton seed companies, while posing a major threat to the entire legal cotton seed market in the country.

To make matters worse, the illegal seeds are sold using the brand name of prominent companies. While the illegal seeds are polluting the environment, the industry is losing legitimate seed sale. There is a dent in tax collections too, Prabhakara Rao, who is also the Managing Director of Nuziveedu Seeds Limited, said.

He lamented that the regulators were focusing only on licensed dealers and seed companies, while unscrupulous players continued to sell the HT seeds without any hindrance.

The focus must be shifted to catching them and taking exemplary and strong punitive action, he said.

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Seed industry raises a flag on surge in HT Bt cotton acreage - BusinessLine