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The recent development of modular universal chimeric antigen receptor (CAR) T-cell platforms that use bifunctional adaptor intermediates to redirect engineered T-cell effector function has greatly expanded the capabilities of adoptive T-cell therapy, enabling safer and more comprehensive cancer treatment. However, universal CAR receptor systems rely on unstable transient recognition of tag-coupled intermediates for T-cell activation, and the array of targeting intermediates has been limited to antibodies and small molecules. Addressing these shortcomings, we engineered universal CAR T-cell receptors that can be covalently modified with synthetic biomaterials in vivo by accelerated SpyCatcher003-SpyTag003 chemistry for cancer-cell targeting. SpyCatcher003-modified CARs, nicknamed DB5 CARs, displayed fast, low-nanomolar reaction kinetics with a synthetic αvβ6-binding peptide that incorporates a SpyTag003 peptide via branched peptide synthesis to comprise a bifunctional intermediate. Prearming DB5 CAR T cells or prelabeling target cells with the bifunctional peptide produced selective CD4 + and CD8 + CAR T-cell responses against αvβ6 + cancer cells in vitro . Furthermore, the synthetic targeting intermediate showed robust DB5 CAR T-cell arming in vivo and selectively reduced αvβ6 + tumor progression in a dual flank xenograft model. We demonstrate the versatility and therapeutic potential of "Cyborg" CAR T-cell therapies that utilize synthetic biomaterials to direct CAR T-cell activity via highly selective bioconjugation that occurs in vivo .

Selective therapeutic targeting of T-cell malignancies is difficult due to the shared lineage between healthy and malignant T cells. Current front-line chemotherapy for these cancers is largely nonspecific, resulting in frequent cases of relapsed/refractory disease. The development of targeting approaches for effectively treating T-cell leukemia and lymphoma thus remains a critical goal for the oncology field. Here, we report the discovery of a DNA aptamer, named HR7A1, that displays low nanomolar affinity for the integrin α4β1 (VLA-4), a marker associated with chemoresistance and relapse in leukemia patients. After truncation of HR7A1 to a minimal binding motif, we demonstrate elevated binding of the aptamer to T-lineage cancer cells over healthy immune cells. Using cryo-EM and competition studies, we find that HR7A1 shares an overlapping binding site on α4β1 with fibronectin and VCAM-1, which has implications for sensitizing blood cancers to chemotherapy. We last characterize barriers to in vivo aptamer translation, including serum stability, temperature-sensitive binding, and short circulation half-life, and synthesize an aptamer-polymer conjugate that addresses these challenges. Future work will seek to validate in vivo targeting of α4β1 + tumors with the conjugate, establishing an aptamer-based biomaterial that can be readily adapted for targeted treatment of T-cell malignancies.

Adult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy., Significance: True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies., (©2024 The Authors; Published by the American Association for Cancer Research.)

The Stimulator of Interferon Genes (STING) pathway is a promising target for cancer immunotherapy. Despite recent advances, therapies targeting the STING pathway are often limited by routes of administration, suboptimal STING activation, or off-target toxicity. Here, we report a dendritic cell (DC)-targeted polymeric prodrug platform (polySTING) that is designed to optimize intracellular delivery of a diamidobenzimidazole (diABZI) small-molecule STING agonist while minimizing off-target toxicity after parenteral administration. PolySTING incorporates mannose targeting ligands as a comonomer, which facilitates its uptake in CD206 + /mannose receptor + professional antigen-presenting cells (APCs) in the tumor microenvironment (TME). The STING agonist is conjugated through a cathepsin B-cleavable valine-alanine (VA) linker for selective intracellular drug release after receptor-mediated endocytosis. When administered intravenously in tumor-bearing mice, polySTING selectively targeted CD206 + /mannose receptor + APCs in the TME, resulting in increased cross-presenting CD8 + DCs, infiltrating CD8 + T cells in the TME as well as maturation across multiple DC subtypes in the tumor-draining lymph node (TDLN). Systemic administration of polySTING slowed tumor growth in a B16-F10 murine melanoma model as well as a 4T1 murine breast cancer model with an acceptable safety profile. Thus, we demonstrate that polySTING delivers STING agonists to professional APCs after systemic administration, generating efficacious DC-driven antitumor immunity with minimal side effects. This new polymeric prodrug platform may offer new opportunities for combining efficient targeted STING agonist delivery with other selective tumor therapeutic strategies., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease., Competing Interests: Declaration of Competing Interest K Lee is a co-founder and employee of GenEdit Inc. Co-authors N. Murthy, S. H. Pun and D. L. Sellers declare no other competing financial interest., (Copyright © 2022. Published by Elsevier B.V.)

Despite the rising global incidence of central nervous system (CNS) disorders, CNS drug development remains challenging, with high costs, long pathways to clinical use and high failure rates. The CNS is highly protected by physiological barriers, in particular, the blood-brain barrier and the blood-cerebrospinal fluid barrier, which limit access of most drugs. Biomaterials can be designed to bypass or traverse these barriers, enabling the controlled delivery of drugs into the CNS. In this Review, we first examine the effects of normal and diseased CNS physiology on drug delivery to the brain and spinal cord. We then discuss CNS drug delivery designs and materials that are administered systemically, directly to the CNS, intranasally or peripherally through intramuscular injections. Finally, we highlight important challenges and opportunities for materials design for drug delivery to the CNS and the anticipated clinical impact of CNS drug delivery., Competing Interests: Competing interests The authors declare no competing interests.

Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell-cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood-brain barrier integrity four days after a controlled cortical impact injury., (© 2021 The Authors.)

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 5×10 5  copies mL -1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics., (© 2021 Wiley-VCH GmbH.)

The integrin αvβ6 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 αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6 + 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 αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 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 αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy., Competing Interests: Conflict of interest M. C. J. has interests in Umoja Biopharma and Juno Therapeutics, a Bristol-Myers Squibb company. M. C. J. is a seed investor and holds ownership equity in Umoja, serves as a member of the Umoja Joint Steering Committee, and is a Board Observer of the Umoja Board of Directors. M. C. J. holds patents, some of which are licensed to Umoja Biopharma and Juno Therapeutics. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects 1-4 . To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examined in vivo , superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

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., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo., Competing Interests: Declaration of Competing Interest A patent application for the VIPER polymer has been filed by the University of Washington. The authors declare no other competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

While biologic drugs such as proteins, peptides, or nucleic acids have shown promise in the treatment of neurodegenerative diseases, the blood-brain barrier (BBB) severely limits drug delivery to the central nervous system (CNS) after systemic administration. Consequently, drug delivery challenges preclude biological drug candidates from the clinical armamentarium. In order to target drug delivery and uptake into to the CNS, we used an in vivo phage display screen to identify peptides able to target drug-uptake by the vast array of neurons of the autonomic nervous system (ANS). Using next-generation sequencing, we identified 21 candidate targeted ANS-to-CNS uptake ligands (TACL) that enriched bacteriophage accumulation and delivered protein-cargo into the CNS after intraperitoneal (IP) administration. The series of TACL peptides were synthesized and tested for their ability to deliver a model enzyme (NeutrAvidin-horseradish peroxidase fusion) to the brain and spinal cord. Three TACL-peptides facilitated significant active enzyme delivery into the CNS, with limited accumulation in off-target organs. Peptide structure and serum stability is increased when internal cysteine residues are cyclized by perfluoroarylation with decafluorobiphenyl, which increased delivery to the CNS further. TACL-peptide was demonstrated to localize in parasympathetic ganglia neurons in addition to neuronal structures in the hindbrain and spinal cord. By targeting uptake into ANS neurons, we demonstrate the potential for TACL-peptides to bypass the blood-brain barrier and deliver a model drug into the brain and spinal cord.

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.

Endocytosed biomacromolecule delivery systems must escape the endosomal trafficking pathway in order for their cargo to exert effects in other cellular compartments. Although endosomal release is well-recognized as one of the greatest barriers to efficacy of biologic drugs with intracellular targets, most drug carriers have relied on cationic materials that passively induce endosomal swelling and membrane rupture with low efficiency. To address the endosome release challenge, our lab has developed a diblock copolymer system for nucleic acid delivery that selectively displays a potent membrane-lytic peptide (melittin) in response to the pH drop during the endosomal maturation. To further optimize this system, we evaluated a panel of peptides with reported lytic activity in comparison to melittin. Nineteen different lytic peptides were synthesized and their membrane-lytic properties at both neutral and acidic pH characterized using a red blood cell hemolysis assay. The top five performing peptides were then conjugated to our pH-sensitive diblock copolymer via disulfide linkers and used to deliver a variety of nucleic acids to cultured mammalian cells as well as in vivo to the mouse brain. We demonstrate that the sharp pH-transition of VIPER compensates for potential advantages from pH-sensitive peptides, such that polymer-peptide conjugates with poorly selective but highly lytic peptides achieve safe and effective transfection both in vitro and in vivo. In addition, peptides that require release from polymer backbones for lysis were less effective in the VIPER system, likely due to limited endosomal reducing power of target cells. Finally, we show that certain peptides are potentiated in lytic ability by polymer conjugation and that these peptide-polymer constructs are most effective in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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.

Cationic polymer gene delivery vehicles that effectively resist premature serum degradation often have difficulty releasing their nucleic acid cargoes. In this work, we report a pH-sensitive polymer (SP), poly(oligo(ethylene glycol) monomethyl ether methacrylate)-co-poly(2-(dimethylamino)ethyl methacrylate)-block- poly(propargyl methacrylate-graft-propyl-(4-methoxy-benzylidene)-amine) (p(PMA-PMBA)-b-(p(OEGMA-DMAEMA)), for successful in vitro and in vivo gene transfer. In the physiological condition, the hydrophobization of p(OEGMA-DMAEMA) polycations by p(PMA-PMBA) significantly enhanced the stability of its polyplexes counterpart. In endosomes, the polymer undergoes an acid-triggered hydrophilic transition through the cleavage of benzoic imines, thus allowing the vector to quickly release nucleic acid cargo due to the loss of hydrophobic functionalization. Compared to a pH-insensitive polymer (IP), SP exhibited more significant luciferase plasmid delivery efficiency with HeLa cells in vitro and with in vivo intraventricular brain injections. Therefore, the polymer designed here is a good solution to address the dilemma of stability and cargo release in gene delivery, and may have broad potential applications in therapeutic agent delivery., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

With an increased prevalence and understanding of central nervous system (CNS) injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the in vivo challenges hindering non-viral nucleic acid delivery to the CNS and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated in vivo gene transfer to the CNS are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for CNS applications and will ultimately bring non-viral vectors closer to clinical application.

Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits further research., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

There is currently no cure for neuron loss in the brain, which can occur due to traumatic injury or neurodegenerative disease. One proposed method to enhance brain neurogenesis is gene transfer to neural progenitor cells. In this work, a guanidine-based copolymer was synthesized and compared to an amine-based copolymer analog previously shown to effectively deliver genes in the murine brain. The guanidine-based copolymer was more efficient at gene transfer to immortalized, cultured cell lines; however, the amine-based copolymer was more effective at gene transfer in the brain. DNA condensation studies revealed that the nucleic acid complexes formed with the guanidine-based copolymer were more susceptible to unpackaging in the presence of anionic proteoglycans compared to complexes formed with the amine-based copolymer. Therefore, polyplexes formed from the amine-based copolymer may be more resistant to destabilization by the heparan sulfate proteoglycans present in the stem cell niches of the brain., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

MMP9-responsive bivalirudin-HPMA copolymers were synthesized for direct, local administration in rat spinal cord contusion injury models. Polymer-conjugated bivalirudin peptides maintained activity while demonstrating enzyme-mediated release upon MMP9 exposure and prolonged release from hyaluronic acid/methylcellulose (HAMC) hydrogels compared to free bivalirudin peptide. Localized administration of bivalirudin copolymers in vivo at the site of rat spinal cord injury decreased cellular proliferation and astrogliosis, suggesting the bivalirudin copolymer and HAMC hydrogel system are a promising therapeutic intervention for reducing immediate inflammatory responses and long term scarring.

Components of the blood have been proposed as potential therapeutic targets for improving cellular regeneration after injury and neurodegenerative disease. In this work, thrombin is shown to increase endogenous neural progenitor proliferation in the intact murine spinal cord. A local injection of heparin before a spinal cord injury reduces cell proliferation and astrogliogenesis associated with scarring. We sought to create depot-formulations of PLGA microsphere and Pluronic F-127 for sustained local delivery of two thrombin inhibitors, heparin and hirudin. Each hydrogel depot-formulation showed delayed drug release compared to microspheres or hydrogel alone. Animals with a lateral demyelination lesion showed a reduction in CD68+ macrophages when treated with hirudin-loaded PLGA/F-127 gels compared to control and heparin-treated animals. Moreover, hirudin-loaded materials showed an accelerated recovery in coordinated stepping and increased oligodendrocyte densities. Together, these data demonstrate that controlled delivery of hirudin accelerates functional recovery from a demyelination lesion in the spinal cord., (Published by Elsevier Ltd.)

Neurological diseases and trauma often cause demyelination, resulting in the disruption of axonal function and integrity. Endogenous remyelination promotes recovery, but the process is not well understood because no method exists to definitively distinguish regenerated from preexisting myelin. To date, remyelinated segments have been defined as anything abnormally short and thin, without empirical data to corroborate these morphological assumptions. To definitively identify regenerated myelin, we used a transgenic mouse with an inducible membrane-bound reporter and targeted Cre recombinase expression to a subset of glial progenitor cells after spinal cord injury, yielding remarkably clear visualization of spontaneously regenerated myelin in vivo. Early after injury, the mean length of sheaths regenerated by Schwann cells and oligodendrocytes (OLs) was significantly shorter than control, uninjured myelin, confirming past assumptions. However, OL-regenerated sheaths elongated progressively over 6 mo to approach control values. Moreover, OL-regenerated myelin thickness was not significantly different from control myelin at most time points after injury. Thus, many newly formed OL sheaths were neither thinner nor shorter than control myelin, vitiating accepted dogmas of what constitutes regenerated myelin. We conclude that remyelination, once thought to be static, is dynamic and elongates independently of axonal growth, in contrast to stretch-based mechanisms proposed in development. Further, without clear identification, past assessments have underestimated the extent and quality of regenerated myelin.

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-l-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendent groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~100-200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1-3, 6-9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.

Progenitors that express NG2-proteoglycan are the predominant self-renewing cells within the CNS. NG2 progenitors replenish oligodendrocyte populations within the intact stem cell niche, and cycling NG2 cells are among the first cells to react to CNS insults. We investigated the role of NG2 progenitors after spinal cord injury and how bone morphogen protein signals remodel the progressive postinjury (PI) niche. Progeny labeled by an NG2-specific reporter virus undergo a coordinated shift in differentiation profile. NG2 progeny born 24 h PI produce scar-forming astrocytes and transient populations of novel phagocytic astrocytes shown to contain denatured myelin within cathepsin-D-labeled endosomes, but NG2 progenitors born 7 d PI differentiate into oligodendrocytes and express myelin on processes that wrap axons. Analysis of spinal cord mRNA shows a temporal shift in the niche transcriptome of ligands that affect PI remodeling and direct progenitor differentiation. We conclude that NG2 progeny are diverse lineages that obey progressive cues after trauma to replenish the injured niche.

Adult neural progenitor cells (aNPCs) exhibit limited migration in vivo with the exception of the rostral migratory stream and injury-induced movement. Surprisingly little is known regarding those signals regulating attraction or inhibition of the aNPC. These studies demonstrate that aNPCs respond principally to a repulsive cue expressed at the embryonic floor plate (FP) and also the injured adult CNS. Adult spinal cord progenitor cells (aSCPs) were seeded onto organotypic slice preparations of the intact embryonic or injured adult spinal cord. Cell migration assays combined with genetic and molecular perturbation of FP-derived migration cues or aSCP receptors establish netrin-1 (Ntn-1) but not Slit-2, Shh, or Ephrin-B3 as the primary FP-derived repellant. When slices were prepared from injured spinal cord, aSCP migration away from the injury core was Ntn-1-dependent. These studies establish Ntn-1 as a critical regulator of aSCP migration in the intact and injured CNS.

Cellullar deficits are replenished within the central nervous system (CNS) by progenitors to maintain integrity and recover function after injury. NG2 proteoglycan-expressing progenitors replenish oligodendrocyte populations, but the nature of NG2 proteoglycan may not indicate a restricted population of progenitors. After injury, restorative spatiotemporal cues have the potential ability to regulate divergent fate-choices for NG2 progenitors, and NG2 progenitors are known to produce multiple cell types in vitro. Recent data suggest that NG2 expression is attenuated while protein levels remain high within injurious tissue; thus, NG2 expression is not static but transiently controlled in response to a dynamic interplay of environmental cues. Therefore, NG2 proteoglycan expression could label newly generated cells or be inherited by resident cell populations that produce oligodendrocytes for remyelination, astrocytes that provide trophic support and other cells that contribute to CNS function.