In vivo selection of synthetic nucleocapsids for tissue targeting.

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

Engineering Low Volume Resuscitants for the Prehospital Care of Severe Hemorrhagic Shock.

Globally, traumatic injury is a leading cause of suffering and death. The ability to curtail damage and ensure survival after major injury requires a time-sensitive response balancing organ perfusion, blood loss, and portability, underscoring the need for novel therapies for the prehospital environment. Currently, there are few options available for damage control resuscitation (DCR) of trauma victims. We hypothesize that synthetic polymers, which are tunable, portable, and stable under austere conditions, can be developed as effective injectable therapies for trauma medicine. In this work, we design injectable polymers for use as low volume resuscitants (LVRs). Using RAFT polymerization, we evaluate the effect of polymer size, architecture, and chemical composition upon both blood coagulation and resuscitation in a rat hemorrhagic shock model. Our therapy is evaluated against a clinically used colloid resuscitant, Hextend. We demonstrate that a radiant star poly(glycerol monomethacrylate) polymer did not interfere with coagulation while successfully correcting metabolic deficit and resuscitating animals from hemorrhagic shock to the desired mean arterial pressure range for DCR - correcting a 60 % total blood volume (TBV) loss when given at only 10 % TBV. This highly portable and non-coagulopathic resuscitant has profound potential for application in trauma medicine.

Design of Polymeric Carriers for Intracellular Peptide Delivery in Oncology Applications.

In recent decades, peptides, which can possess high potency, excellent selectivity, and low toxicity, have emerged as promising therapeutics for cancer applications. Combined with an improved understanding of tumor biology and immuno-oncology, peptides have demonstrated robust antitumor efficacy in preclinical tumor models. However, the translation of peptides with intracellular targets into clinical therapies has been severely hindered by limitations in their intrinsic structure, such as low systemic stability, rapid clearance, and poor membrane permeability, that impede intracellular delivery. In this Review, we summarize recent advances in polymer-mediated intracellular delivery of peptides for cancer therapy, including both therapeutic peptides and peptide antigens. We highlight strategies to engineer polymeric materials to increase peptide delivery efficiency, especially cytosolic delivery, which plays a crucial role in potentiating peptide-based therapies. Finally, we discuss future opportunities for peptides in cancer treatment, with an emphasis on the design of polymer nanocarriers for optimized peptide delivery.

Evolution of a designed protein assembly encapsulating its own RNA genome.

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

Optimized serum stability and specificity of an αvß6 integrin-binding peptide for tumor targeting.

The integrin αvß6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvß6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvß6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvß6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvß6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvß6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvß6 in vivo, expanding opportunities for cancer targeting and therapy.

Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

SCORe: SARS-CoV-2 Omicron Variant RBD-Binding DNA Aptamer for Multiplexed Rapid Detection and Pseudovirus Neutralization.

During the COVID-19 (coronavirus disease 2019) pandemic, several SARS-CoV-2 variants of concern emerged, including the Omicron variant, which has enhanced infectivity and immune invasion. Many antibodies and aptamers that bind the spike (S) of previous strains of SARS-CoV-2 either do not bind or bind with low affinity to Omicron S. In this study, we report a high-affinity SARS-CoV-2 Omicron RBD-binding aptamer (SCORe) that binds Omicron BA.1 and BA.2 RBD with nanomolar KD1. We employ aptamers SCORe.50 and SNAP4.74 in a multiplexed lateral flow assay (LFA) to distinguish between Omicron and wild-type S at concentrations as low as 100 pM. Finally, we show that SCORe.50 and its dimerized form SCOReD can neutralize Omicron S-pseudotyped virus infection of ACE2-overexpressing cells by >70%. SCORe therefore has potential applications in COVID-19 rapid diagnostics as well as in viral neutralization.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery.

Despite remarkable advances over the past several decades, many therapeutic nanomaterials fail to overcome major in vivo delivery barriers. Controlling immunogenicity, optimizing biodistribution, and engineering environmental responsiveness are key outstanding delivery problems for most nanotherapeutics. However, notable exceptions exist including some lipid and polymeric nanoparticles, some virus-based nanoparticles, and nanoparticle vaccines where immunogenicity is desired. Self-assembling protein nanoparticles offer a powerful blend of modularity and precise designability to the field, and have the potential to solve many of the major barriers to delivery. In this review, we provide a brief overview of key designable features of protein nanoparticles and their implications for therapeutic delivery applications. We anticipate that protein nanoparticles will rapidly grow in their prevalence and impact as clinically relevant delivery platforms.

Biomaterials in Chimeric Antigen Receptor T-Cell Process Development.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivo modified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy's broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.Recognizing that clinical T-cell isolation reagents have high cost and supply constraints, we developed a synthetic DNA aptamer and complementary reversal agent technology that isolates label-free CD8+ T cells with high purity and yield from peripheral blood mononuclear cells. Encouragingly, CAR T cells manufactured from both antibody- and aptamer-isolated T cells were comparable in therapeutic potency. Discovery and design of other T-cell specific aptamers and corresponding reversal reagents could fully realize the potential of this approach, enabling inexpensive isolation of multiple distinct T-cell populations in a single isolation step.Current ex vivo T-cell activation materials do not accurately mimic in situ T-cell activation by antigen presenting cells (APCs). They cause unequal CD4+ and CD8+ T-cell expansion, necessitating separate production of CD4+ and CD8+ CAR T cells for therapies that call for balanced infusion compositions. To address these shortcomings, we designed a panel of biodegradable cell-templated silica microparticles with supported lipid bilayers that display stimulatory ligands for T-cell activation. High membrane fluidity, elongated shape, and rough surface topography, all properties of endogenous APCs, were found to be favorable parameters for activation, promoting unbiased and efficient CD4/CD8 T-cell expansion while not terminally differentiating the cells.Viral and electroporation-based gene delivery systems have various drawbacks. Viral vectors are expensive and have limited cargo sizes, whereas electroporation is highly cytotoxic. Thus, low-cost nonviral platforms that transfect T cells with low cytotoxicity and high efficiency are needed for CAR gene delivery. Our group thus synthesized a panel of cationic polymers with different architectures and evaluated their T-cell transfection ability. We identified a comb-shaped polymer formulation that transfected primary T cells with low cytotoxicity, although transfection efficiency was low compared to conventional methods. Analysis of intracellular and extracellular barriers to transfection revealed low uptake of polyplexes and high endosomal pH in T cells, alluding to biological and polymer properties that could be further improved.These innovations represent just a few recent developments in the biomaterials field for addressing CAR T-cell production needs. Together, these technologies and their future advancement will pave the way for economical and straightforward CAR T-cell manufacturing.

Dual-Stimuli Responsive Single-Chain Polymer Folding via Intrachain Complexation of Tetramethoxyazobenzene and ß-Cyclodextrin.

We synthesize and characterize a triblock polymer with asymmetric tetramethoxyazobenzene (TMAB) and ß-cyclodextrin functionalization, taking advantage of the well-characterized azobenzene derivative-cyclodextrin inclusion complex to promote photoresponsive, self-contained folding of the polymer in an aqueous system. We use 1H NMR to show the reversibility of (E)-to-(Z) and (Z)-to-(E) TMAB photoisomerization, and evaluate the thermal stability of (Z)-TMAB and the comparatively rapid acid-catalyzed thermal (Z)-to-(E) isomerization. Important for its potential use as a functional material, we show the photoisomerization cyclability of the polymeric TMAB chromophore and calculate isomerization quantum yields by extinction spectroscopy. To verify self-inclusion of the polymeric TMAB and cyclodextrin, we use two-dimensional 1H NOESY NMR data to show proximity of TMAB and cyclodextrin in the (E)-state only; however, (Z)-TMAB is not locally correlated with cyclodextrin. Finally, the observed decrease in photoisomerization quantum yield for the dual-functionalized polymer compared to the isolated chromophore in an aqueous solution confirms TMAB and ß-cyclodextrin not only are in proximity to one another, but also form the inclusion complex.

Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection.

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.

Identification of a DNA Aptamer That Binds to Human Monocytes and Macrophages.

As cancer strategies shift toward immunotherapy, the need for new binding ligands to target and isolate specific immune cell populations has soared. Based on prior work identifying a peptide specific for murine M2-like macrophages, we sought to identify an aptamer that could bind human M2-like macrophages. Tumor-associated macrophages (TAMs) adopt an M2-like phenotype and support tumor progression and dissemination. Here, we employed cell-SELEX to identify an aptamer ligand that targets this cell population over tissue resident (M0-like) or tumoricidal (M1-like) macrophages. Instead, we identified an aptamer that binds both human M0- and M2-like macrophages and monocytes, with highest binding affinity to M2-like macrophage (Kd ∼ 20 nM) and monocytes (Kd ∼ 45 nM) and minimal binding to other leukocytes. The aptamer binds to CD14+ but not CD16+ monocytes, and is rapidly internalized by these cells. We also demonstrate that this aptamer is able to bind human monocytes when both are administered in vivo to mice. Thus, binding to these cell populations (monocytes, M0-like and M2-like macrophages), this aptamer lends itself toward monocyte-specific applications, such as monocyte-targeted drug delivery or column selection.

Formulation of thrombin-inhibiting hydrogels via self-assembly of ionic peptides with peptide-modified polymers.

Cell therapy for spinal cord injuries offers the possibility of replacing lost cells after trauma to the central nervous system (CNS). In preclinical studies, synthetic hydrogels are often co-delivered to the injury site to support survival and integration of the transplanted cells. These hydrogels ideally mimic the mechanical and biochemical features of a healthy CNS extracellular matrix while also providing the possibility of localized drug delivery to promote healing. In this work, we synthesize peptide-functionalized polymers that contain both a peptide sequence for incorporation into self-assembled peptide hydrogels along with bioactive peptides that inhibit scar formation. We demonstrate that peptide hydrogels formulated with the peptide-functionalized polymers possess similar mechanical properties (soft and shear-thinning) as peptide-only hydrogels. Small angle neutron scattering analysis reveals that polymer-containing hydrogels possess larger inhomogeneous domains but small-scale features such as mesh size remain the same as peptide-only hydrogels. We further confirm that the integrated hydrogels containing bioactive peptides exhibit thrombin inhibition activity, which has previously shown to reduce scar formation in vivo. Finally, while the survival of encapsulated cells was poor, cells cultured on the hydrogels exhibited good viability. Overall, the described composite hydrogels formed from self-assembling peptides and peptide-modified polymers are promising, user-friendly materials for CNS applications in regeneration.

Synthesis and Characterization of Anionic Poly(cyclopentadienylene vinylene) and Its Use in Conductive Hydrogels.

The use of π-conjugated polymers (CPs) in conductive hydrogels remains challenging due to the water-insoluble nature of most CPs. Conjugated polyelectrolytes (CPEs) are promising alternatives because they have tunable electronic properties and high water-solubility, but they are often difficult to synthesize and thus have not been widely adopted. Herein, we report the synthesis of an anionic poly(cyclopentadienylene vinylene) (aPCPV) from an insulating precursor under mild conditions and in high yield. Functionalized aPCPV is a highly water-soluble CPE that exhibits low cytotoxicity, and we found that doping hydrogels with aPCPV imparts conductivity. We also anticipate that this synthetic strategy, due to its ease and high efficiency, will be widely used to create families of not-yet-explored π-conjugated vinylene polymers.

pH-Sensitive Polymers as Dynamic Mediators of Barriers to Nucleic Acid Delivery.

The nonviral delivery of exogenous nucleic acids (NA) into cells for therapeutic purposes has rapidly matured into tangible clinical impact. Synthetic polymers are particularly attractive vectors for NA delivery due to their relatively inexpensive production compared to viral alternatives and their highly tailorable chemical properties; indeed, many preclinical investigations have revealed the primary biological barriers to nonviral NA delivery by systematically varying polymeric material properties. This review focuses on applications of pH-sensitive chemistries that enable polymeric vectors to serially address multiple biological barriers to NA delivery. In particular, we focus on recent innovations with in vivo evaluation that dynamically enable colloidal stability, cellular uptake, endosomal escape, and nucleic acid release. We conclude with a summary of successes to date and projected areas for impactful future research.

Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration.

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

Serum Nuclease Susceptibility of mRNA Cargo in Condensed Polyplexes.

Messenger RNA (mRNA) is a biomolecule with a wide range of promising clinical applications. However, the unstable nature of mRNA and its susceptibility to degradation by ribonucleases (RNases) necessitate the use of specialized formulations for delivery. Polycations are an emerging class of synthetic carriers capable of packaging nucleic acids, and may serve as suitable RNase-resistant formulations for mRNA administration. Here, we explore the application of VIPER and sunflower polycations, two polycations previously synthesized by our group, for the delivery of mRNA in comparison to branched poly(ethylenimine); all three polycations have been shown to efficiently deliver plasmid DNA (pDNA) to cultured cells. Despite successful mRNA condensation and packaging, transfection studies reveal that these three polycations can only efficiently deliver mRNA under serum-free conditions, while pDNA delivery is achieved even in the presence of serum. RNase resistance studies confirm that nuclease degradation of mRNA cargo remains a significant barrier to mRNA delivery using these polycations. These results emphasize the need for additional strategies for nuclease protection of mRNA cargo beyond electrostatic complexation with polycation.

A Facile Cyclization Method Improves Peptide Serum Stability and Confers Intrinsic Fluorescence.

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F-2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F-2CN cyclization of an M2 macrophage-targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole-organ fluorescence imaging and cell internalization by flow cytometry.

Tunable, Injectable Hydrogels Based on Peptide-Cross-Linked, Cyclized Polymer Nanoparticles for Neural Progenitor Cell Delivery.

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.