Estimating population size is essential for many applications in population ecology, so capture–recapture techniques to do this are often taught in secondary school classrooms and introductory university units. However, few classroom simulations of capture–recapture consider the sensitivity of results to sampling intensity, the important concept that the population size calculated is an estimate with error attached, or the consequences of violating assumptions underpinning particular capture–recapture models. We describe a simple approach to teaching the Lincoln index method of capture–recapture using packs of playing cards. Students can trial different sampling intensities, calculate 95% confidence intervals for population estimates, and explore the consequences of violating specific assumptions.
<p>Supplementary Figure S2. OX40L, CD80 and CD86 mRNA-CART treatment induce long lasting protection against re-challenge with the same tumor cell line. Supplementary Figure S3. OX40L, CD80 and CD86 mRNA-CART treatment do not result in long term elevated levels of pro-inflammatory cytokines in serum of treated mice.</p>
Abstract Localized expression of effector molecules can initiate antitumor responses through engagement of specific receptors on target cells in the tumor microenvironment. These locally induced responses may also have a systemic effect, clearing additional tumors throughout the body. In this study, to evoke systemic antitumor responses, we utilized charge-altering releasable transporters (CART) for local intratumoral delivery of mRNA coding for costimulatory and immune-modulating factors. Intratumoral injection of the CART–mRNA complexes resulted in mRNA expression at the site of administration, transfecting a substantial proportion of tumor-infiltrating dendritic cells, macrophages, and T cells in addition to the tumor cells, resulting in a local antitumor effect. Using a two-tumor model, we further show that mRNA therapy locally administered to one tumor stimulated a systemic antitumor response, curing both tumors. The combination of Ox40l-, Cd80-, and Cd86-encoding mRNA resulted in the local upregulation of proinflammatory cytokines, robust local T-cell activation, and migration of immune cells to local draining lymph node or to an anatomically distant tumor. This approach delayed tumor growth, facilitated tumor regression, and cured tumors in both A20 and CT26 tumor models. These results highlight mRNA-CART therapy as a viable approach to induce systemic antitumor immunity from a single localized injection. Significance: The mRNA-CART system is a highly effective delivery platform for delivering immunostimulatory genes into the tumor microenvironment for potential therapeutic development.
The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with lipid nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called charge-altering releasable transporters (CARTs). Using these inherently nonimmunogenic vehicles, we can tailor the vaccine immunogenicity by inclusion of coformulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of receptor binding domain (RBD)-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long-lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.
Abstract The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific T H 1 T cell responses including CD4 + and CD8 + T cell memory.
Significance Protein expression using mRNA has the potential to transform many areas of life science research and affect the prevention, detection, and treatment of disease. However, realizing this potential requires the development of readily accessible, efficacious, and safe delivery systems that can functionally deliver mRNA to cells in culture and in vivo. A class of materials developed for mRNA delivery is described that operates through an unprecedented self-immolation mechanism. These materials are accessed in two steps through an organocatalytic oligomerization. They noncovalently complex, protect, deliver, and release mRNA with >99% transfection efficiency in cultured cells and with robust protein expression in mice using multiple routes of administration. This mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.
Significance The RNA delivery field is mostly focused on lipid nanoparticles (LNPs). Although promising, LNPs have several limitations with respect to pharmacokinetics, biodistribution, and toxicity. The mechanism of RNA charge-altering releasable transporters (CART) delivery and release is unique. It proceeds dynamically with a controllable change in physical properties. Differing from all mRNA delivery systems, a key attribute of CARTs is a charge-altering degradation mechanism, which transforms the initial polycationic CART into neutral byproducts, thereby enabling endosomal escape, release, and subsequent translation of the polyanionic mRNA cargo. With this study, we introduce a potentially general approach to therapeutic vaccination enabled by a dynamic drug-delivery system (mRNA-CART) and demonstrate its utility in suppressing tumor formation and in eliminating established tumors.
We report the development of post-transcriptional chemical methods that enable control over CRISPR-Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N3) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated ("cloaked") sgRNA was completely inactive in DNA cleavage with Cas9 in vitro, and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR-Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.
<div>Abstract<p>Localized expression of effector molecules can initiate antitumor responses through engagement of specific receptors on target cells in the tumor microenvironment. These locally induced responses may also have a systemic effect, clearing additional tumors throughout the body. In this study, to evoke systemic antitumor responses, we utilized charge-altering releasable transporters (CART) for local intratumoral delivery of mRNA coding for costimulatory and immune-modulating factors. Intratumoral injection of the CART–mRNA complexes resulted in mRNA expression at the site of administration, transfecting a substantial proportion of tumor-infiltrating dendritic cells, macrophages, and T cells in addition to the tumor cells, resulting in a local antitumor effect. Using a two-tumor model, we further show that mRNA therapy locally administered to one tumor stimulated a systemic antitumor response, curing both tumors. The combination of <i>Ox40l</i>-, <i>Cd80</i>-, and <i>Cd86</i>-encoding mRNA resulted in the local upregulation of proinflammatory cytokines, robust local T-cell activation, and migration of immune cells to local draining lymph node or to an anatomically distant tumor. This approach delayed tumor growth, facilitated tumor regression, and cured tumors in both A20 and CT26 tumor models. These results highlight mRNA-CART therapy as a viable approach to induce systemic antitumor immunity from a single localized injection.</p>Significance:<p>The mRNA-CART system is a highly effective delivery platform for delivering immunostimulatory genes into the tumor microenvironment for potential therapeutic development.</p></div>
The oxidative lactonization of N-substituted diethanolamines with the Pd catalyst [LPd(OAc)]22+[OTf–]2 generates N-substituted morpholin-2-ones. The organocatalytic ring-opening polymerization of N-acyl morpholin-2-ones occurs readily to generate functionalized poly(aminoesters) with N-acylated amines in the polyester backbone. The thermodynamics of the ring-opening polymerization depends sensitively on the hybridization of the nitrogen of the heterocyclic lactone. N-Acyl morpholin-2-ones polymerize readily to generate polymorpholinones, but the N-aryl or N-alkyl substituted morpholin-2-ones do not polymerize. Experimental and theoretical studies reveal that the thermodynamics of ring opening correlates to the degree of pyramidalization of the endocyclic N-atom. Deprotection of the poly(N-Boc-morpholin-2-one) yields a water-soluble, cationic polymorpholinone.
