Can nanoparticles be used to deliver the CRISPR-Cas system for gene editing?

Written by: Abby Stahl

Original Article: Liu et al. Advanced Materials 2019
The Gist of It
You may have heard of the CRISPR-Cas system for genome editing before. Using molecules named “Cas proteins” and “single guide RNAs” (sgRNAs), scientists can selectively cut a single, specific spot in DNA. This is a handy tool to delete parts of DNA that are no longer wanted or needed. For example, removing the gene PCSK9, which encodes a protein that controls cholesterol levels in your blood, can have benefits for heart health, and deleting the gene HPV18, which is known to be associated with cervical cancer, can reduce human cervical cancer growth. Great! The catch is that getting these DNA editing tools into cells is tricky. Nanoparticles, which are tiny shells composed of proteins, oils, or fats, can be loaded with the DNA editing molecules and taken up by cells. Dr. Liu and colleagues have designed new types of nanoparticles that can deliver CRISPR molecules into cells both in culture dishes and even in living animals. The scientists made a unique formulation of nanoparticles using a fat called BAMEA-O16B, which has added sulfur molecules to create disulfide bonds that allow the particles to break down inside cells and release their cargo. First, the scientists showed that the BAMEA-O16B nanoparticles could shuttle RNA molecules better than nanoparticles without these disulfide bonds, or RNA alone, as a proof-of-concept for delivery. Then the team effectively delivered CRISPR molecules into cells using the BAMEA-O16B nanoparticles, leading to the deletion of the targeted gene in 90% of the cells. When these CRISPR-Cas nanoparticles were used to remove the HPV18 gene from cells growing in culture dishes, they stopped the growth of human cervical cancer cells. Excitingly, when they were used to remove the PCSK9 gene in mice, the CRISPR-Cas nanoparticles reduced the amount of PCSK9 protein in the serum of mice. This work establishes a new and exciting way to deliver the CRISPR-Cas system that could be used to treat cancer and reduce cardiovascular disease in the future.
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Nanoparticles composed of BAMEA-O16B deliver Cas9 and single guide RNA (sgRNA) into cells in culture dishes and in organisms.

The Nitty Gritty
Optimizing the delivery of therapeutic proteins and RNA molecules is an active area of research for many fields, including CRISPR-Cas–mediated gene editing. Viral vectors have historically been used for delivery, but they are not ideal due to cargo size restrictions and immunogenicity. Nanoparticles offer one potential solution to this delivery challenge. In this work, the authors synthesized particles from hydrophobic lipid tails containing disulfide bonds. The leading formulation, termed BAMEA-O16B, encapsulates mRNA by electrostatic interactions and is degraded in reductive chemical environments to release the mRNA. Mechanistically, removing the disulfide bonds restricts mRNA release from the endosome by four-fold; thus, the disulfide bonds are critical for bioreduction and degradation of the nanoparticles. Using this formulation, the authors showed that BAMEA-O16B has reduced cytotoxicity compared to a standard transfection agent, Lipofectamine 2000 (LPF2K), in human A375 cells by measuring cell death. A dose of 160 ng/mL BAMEA-O16B loaded with red fluorescent protein (RFP)-encoding mRNA resulted in 90% of HeLa cells expressing RFP, demonstrating a proof-of-concept for mRNA delivery. Next, the authors loaded Cas9 mRNA and sgRNA into BAMEA-O16B nanoparticles. The nanoparticles (≈ 230 nm) maintained electrostatic interactions even with this substantially larger cargo. When loaded with a sgRNA designed to delete GFP from stably-expressing HEK cells, BAMEA-O16B nanoparticles were less cytotoxic than LPF2K, with > 90% of cells maintaining viability. As the concentration of Cas9 mRNA was increased from 20 to 160 ng/mL, the GFP knockout efficiency increased from 35% to 90%, peaking as early as 36 hours post-delivery. To demonstrate the use of BAMEA-O16B nanoparticles for therapeutic gene editing, the authors designed sgRNAs to inactivate the human papillomavirus type 18 (HPV18) gene in human cervical cancer cells. A dose of 320 ng/mL Cas9 mRNA and sgRNA reduced HeLa cell viability to 30%, while a scrambled sgRNA control had no effect. Finally, the authors delivered their BAMEA-O16B nanoparticles containing a sgRNA targeting PCSK9 into mice intravenously. Cas9 and sgRNA mainly accumulated in the liver after injection, and serum PCSK9 levels were reduced by 80%, while injections of PBS and scrambled sgRNA control had no effect. Furthermore, there was no liver inflammation or hepatocellular injury histologically, and minimal changes in secreted liver factors such as serum aspartate transaminase, alanine aminotransferase, and total bilirubin occurred. The authors suggest that the knockdown of PCSK9 will be a clinically meaningful application of CRISPR-Cas editing with some existing regulatory predicates on the pathway to clinical translation.
Original Research Article: Liu J., et al. “Fast and efficient CRISPR/Cas9 genome editing in vivo enabled by bioreducible lipid and messenger RNA nanoparticles.Adv Mater 31.33 (2019): e1902575. doi: 10.1002/adma.201902575

What holds back our immune system from attacking our bodies?

Written By: Kaitlyn Sadtler

Original Article: Moser et al. Journal of Experimental Medicine 2019
The Gist of It:
Our body has amazing capabilities to defend itself from danger. Our immune cells fight off countless potential pathogens every day, while at the same time preventing the destruction of our own cells. However, when things go awry, we can see different illnesses that can broadly be defined as “autoimmune diseases”. These diseases have a variety of causes, including genetic, viral, or idiopathic (a fancy word that doctors use instead of “we don’t know”). The more we know about how our body naturally prevents autoimmune diseases, the more we can learn about how things may be unbalanced in patients with autoimmune conditions. Recent work from the Children’s Hospital of Philadelphia has shed some light on how our antibody-producing cells (called B cells) regulate themselves. B cells, named for the bursa of Fabricus (an organ in the chicken where B cells were originally discovered – your fun immunology fact of the day!), are stimulated by antigens, which are small pieces of proteins that, like barcodes on food at the grocery store, help the B cell identify the piece of protein and, if it identifies the piece of protein as foreign (aka not belonging to your body), produce the proper antibodies to neutralize the threat. In autoimmune diseases, B cells can start to produce antibodies that attack our body’s own proteins instead. One protein inside our cells that is called “Itch” controls the activation of B cells. Mice that lacked the protein Itch had higher numbers of activated B cells, which had increased sugar metabolism and energy consumption. Understanding these mechanisms of how our body keeps itself in check will help scientists develop new treatments to curb autoimmune diseases in a targeted manner as opposed to simply using broad-spectrum steroids, which is the current standard practice but does not impact the underlying problems to actually solve the problem causing the disease.
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The protein Itch helps prevent autoimmunity by regulating B cell activation and responses.

The Nitty Gritty:
Previous research has shown that Itch (an E3 ubiquitin ligase) can regulate autoimmunity. Itch knockout (KO) mice show autoimmune symptoms including the presence of autoantibodies (anti-dsDNA IgG). Moser et al. showed that in Itch KO mice, there were larger proportions of germinal center B cells (GL7+CD38) and class switched non-germinal center B cells (GL7) in the spleen and higher proportions of class-switched IgM and IgG1+ plasma cells in the bone marrow (BM). In a mixed BM chimera (using CD45.1 WT and CD42.2 Itch KO BM) there was a higher proportion of CD45.2+ Itch KO B cells. Through proteomic analysis of WT and Itch KO B cells that were stimulated with CpG, the authors showed higher expression of proteins associated with mTORC1 activation, cell cycle & E2F transcription factors as well as G2/M checkpoint proteins, and proteins associated with inflammatory responses to IFNγ and TNF. In accordance with the upregulation of cell cycle-associated proteins, Itch KO B cells had increased cell division in vitro when stimulated with anti-IgM or CpG when compared with a WT control (assessed via staining with a cell tracker dye). As expected based on the mTORC1 activation signature, Itch KO mice also displayed heightened glycolysis, which was determined by an increase in the extracellular acidification rate after stimulation with oligomycin in a Seahorse assay. After immunizing mice with NP-OVA, there were greater numbers of B18 germinal center B cells and NP+ plasma cells. The antibodies produced by this response were of higher affinity in the KO mouse than in the WT and displayed more mutation in the CDR1 gene region. Furthermore, when stained with a cell tracker dye and transferred in vivo, Itch KO B cells proliferated more after immunization and expressed higher levels of P-S6 (phosphorylated-S6) after a 1-hour ex vivo culture compared with WT B cells, suggesting Itch limits both cell division and mTORC1 activation.
Moser E.K., et al. “The E3 ubiquitin ligase Itch restricts antigen-driven B cell responses.” J Exp Med (2019): pii: jem.20181953.

I’m so hungry my stomach is eating itself: how a pathogen uses autophagy to be successful

Written By: Rebecca Tweedell

Original Article: Shimamura et al. Frontiers in Microbiology 2019
The Gist of It:
In people with healthy, fully functional immune systems, we don’t often hear about fungal infections. However, in people who are immunocompromised, including infants, the elderly, patients undergoing chemotherapy, and many others, fungal infections are much more common. One of these opportunistic fungal pathogens is Candida glabrata. The incidence of C. glabrata infections increased four-fold between 1992–1993 and 2008–2011, but little is known about how it achieves its virulence. One thing that is known is that fungi can use a process called autophagy to help them survive when nutrients are lacking, and some believe that this process may also be important for virulence. The word “autophagy” comes from “auto”, meaning “self”, and “phagein”, meaning “eat”; so autophagy = self eat. Kind of like in that saying “I’m so hungry my stomach is eating itself!”, during autophagy, cells break down some of their internal contents to recycle their building blocks to make new things that they need. Researchers at Nagasaki University have been studying how autophagy might play a role in C. glabrata’s virulence. They compared normal C. glabrata to a mutated strain that could not perform autophagy and found that the mutated strain did not grow as quickly under nutrient-limiting conditions. Additionally, this mutant did not handle other stresses, such as treatment with hydrogen peroxide, as well as normal fungi that could use autophagy did. Most importantly, in mouse models of C. glabrata infection, they found that the fungi that could not use autophagy did not infect mice as well as normal fungi did. This research highlights the importance of autophagy in not only the pathogen’s survival during stress, but also its ability to be a successful pathogen. Based on this work, treatments that block autophagy may be successful in treating C. glabrata infections, reducing the threat of these pathogens to those at risk.
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C. glabrata that cannot recycle their components through autophagy are more likely to die under stress conditions and cannot infect well.

The Nitty Gritty:
A C. glabrata strain that could not perform autophagy was created by deleting Atg1, a gene that encodes a protein in the ATG protein complex required for the induction of autophagy. The Cgatg1Δ strain was compared to the wild type (WT) and to a reconstituted strain that had ATG1 expression restored (Cgatg1Δ + CgATG1). Using spot assays in which the same number of cells from each strain was spotted on agar plates, researchers found that the Cgatg1Δ strain had growth defects in the presence of H2O2 and under nitrogen starvation conditions, while Cgatg1Δ + CgATG1 growth was comparable to that of WT. When treated with H2O2, the Cgatg1Δ cells had 60-fold higher levels of reactive oxygen species compared with WT and the Cgatg1Δ + CgATG1 strain, which likely contributed to its growth defect. In an ex vivo experiment, fungi were co-incubated with murine peritoneal macrophages, and the growth of the fungi was monitored. While all strains could be phagocytosed by the macrophages, the WT and Cgatg1Δ + CgATG1 strain were able to continue to grow successfully despite the phagocytosis, while the Cgatg1Δ strain could not. Finally, BALB/c mice were infected with C. glabrata in two different models to monitor the effect of autophagy on virulence. In the disseminated candidiasis model (C. glabrata injected intravenously), the Cgatg1Δ strain had fewer colony forming units (CFU) per organ in the liver and spleen but comparable numbers in the kidney when compared with WT. In the intra-abdominal candidiasis model (C. glabrata injected intra-abdominally), the Cgatg1Δ strain had fewer CFU per organ in the liver, spleen, and pancreas. Taken together, these results show that autophagy is critical for C. glabrata survival under nutrient-deficient and stress conditions, and autophagy plays a role in fitness/viability during infection.
Original Research Article: Shimamura, S., et al. “Autophagy-inducing factor Atg1 is required for virulence in the pathogenic fungus Candida glabrata.” Front Microbiol 10 (2019): 27.

Understanding the mechanisms of fertility

Written By: Kaitlyn Sadtler

Original Article: Saatcioglu et al. eLife 2019
The Gist of It:
Development of the reproductive system in humans involves a complex set of biologic systems and begins in the womb within 1 month after fertilization of the egg. In XX females (who will typically develop a uterus, ovaries, and vagina) and XY males (who will typically develop testes, seminal vesicles, and a penis), the differentiation between these two sets of body parts begins just a few weeks later. After being born, there are further maturation events that must occur during puberty to form functional reproductive organs. Clearly, there are many, many steps in sexual development, and errors in several of these steps can lead to a reproductive system that doesn’t function quite right, causing infertility. To help understand the causes of infertility and hopefully develop new therapeutics, researchers are studying multiple steps in these developmental pathways. Recently, scientists from Mass General Hospital used single-cell sequencing to analyze the formation of endometrial tissue (the lining of the uterus). They discovered a specific cell type that expresses a gene called Misr2+ that is required for the formation of a functional endometrium. They injected the protein MIS, which kills cells with Misr2, into female rats and mice at different times after birth. Injection of MIS early after birth, but not later, resulted in the death of Misr2+ cells and decreased the fertility of the mice. Furthermore, when comparing genes associated with the resulting stunted cell population in the mice with genes from humans with Mullerian aplasia or hypoplasia (XX females with alterations in the development of internal genitalia), the researchers noted several genes that were common between the two groups, suggesting a potential link between Misr2+ cell development and human uterine development. Further research on this topic will allow us to understand more of the intricacies of sexual development and how we could develop therapeutics to help promote fertility.
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Misr2+ Cells found in the Mullerian Subluminal Mesenchyme are required for fertility — expression of MIS in XX females results in uterine hypoplasia and infertility.

The Nitty Gritty:
Using mouse and rat models, Saatcioglu et al. analyzed the role of Misr2+ cells of the postnatal uterus and their effect on downstream fertility. The authors identified Misr2+ cells in the female Mullerian subluminal mesenchyme via Misr2-CRE/TdTomato lineage tracing and RNA in situ hybridization. MIS (Mullerian inhibitory protein), which is expressed by XY males, induces regression of Mullerian ducts to allow for development of male reproductive organs. MIS induces apoptosis in the Misr2+ cells in males while these cells persist in females (in the absence of MIS). Following administration of MIS via adeno-associated virus (AAV) delivery at post-natal day 1 (PND1), there was an alteration of uterine morphology by PND6 and severe uterine hypoplasia by PND20. This MIS treatment resulted in a population of cells that the researchers described as “inhibited progenitors” that they further evaluated with single-cell RNA sequencing. By sequencing cells from the PND6 uterus, the authors characterized the following populations: outer stroma, inner stroma, myometrium, luminal epithelium, dividing mesenchyme, vascular endothelium, dividing epithelium, mesothelial, myeloid, erythroid, pericyte, lymphatic endothelium, and nerve cells. When comparing the MIS-treated uterus with the control, the inhibited progenitor cell population comprised roughly 10% of the cells that were analyzed. When compared with gene expression patterns in humans with Mullerian aplasia, several genes were enriched in both the human disease and the inhibited progenitor cluster, including Khdrbs2, Gfra1, Gata3, and Fgfr2. Loss of Misr2+ progenitors resulted in decreased fertility in rodent models, but these cells were only required for proper development and fertility during the first 6 days after birth. These MISR2+ cells were also present in the developing human reproductive tract and were detected in archival samples from 22 to 37 weeks of gestation.
Original Research Article: Saatcioglu, H.D., et al. “Single-cell sequencing of neonatal uterus reveals an Misr2+ endometrial progenitor indispensable for fertility.” eLife 8 (2019): e46349.