Six Stanford University researchers are among the 120 newly elected members of the National Academy of Sciences. Scientists are elected to the NAS by their peers.

The six Stanford faculty members newly elected to the National Academy of Sciences. (Image credit: Andrew Brodhead)

The new members from Stanford are Savas Dimopoulos, the Hamamoto Family Professor and professor of physics in the School of Humanities and Sciences; Daniel Freedman, a visiting professor at theStanford Institute for Theoretical Physics (SITP) and professor of applied mathematics and theoretical physics, emeritus, at MIT; Judith Frydman, professor of biology and the Donald Kennedy Chair in the School of Humanities and Sciences, and professor of genetics in the Stanford School of Medicine; Kathryn A. Kam Moler, vice provost and dean of research, and the Marvin Chodorow Professor and professor of applied physics and of physics in the School of Humanities and Sciences; Tirin Moore, professor of neurobiology in the Stanford School of Medicine; and John Rickford, professor of linguistics and the J.E. Wallace Sterling Professor in the Humanities, emeritus, in the School of Humanities and Sciences.

Savas Dimopoulos collaborates on a number of experiments that use the dramatic advances in atom interferometry to do fundamental physics. These include testing Einsteins theory of general relativity to fifteen decimal precision, atom neutrality to thirty decimals, and looking for modifications of quantum mechanics. He is also designing an atom-interferometric gravity-wave detector that will allow us to look at the universe with gravity waves instead of light.

Daniel Freedmans research is in quantum field theory, quantum gravity and string theory with an emphasis on the role of supersymmetry. Freedman, along with physicists Sergio Ferrara and Peter van Nieuwenhuizen, developed the theory of supergravity. A combination of the principles of supersymmetry and general relatively, supergravity is a deeply influential blueprint for unifying all of natures fundamental interactions.

Judith Frydman uses a multidisciplinary approach to address fundamental questions about protein folding and degradation, and molecular chaperones, which help facilitate protein folding. In addition, this work aims to define how impairment of cellular folding and quality control are linked to disease, including cancer and neurodegenerative diseases, and examine whether reengineering chaperone networks can provide therapeutic strategies.

Kam Molers research involves developing new tools to measure magnetic properties of quantum materials and devices on micron length-scales. These tools can then be used to investigate fundamental materials physics, superconducting devices and exotic Josephson effects a phenomenon in superconductors that shows promise for quantum computing.

Tirin Moore studies the activity of single neurons and populations of neurons in areas of the brain that relate to visual and motor functions. His lab explores the consequences of changes in that activity and aims to develop innovative approaches to fundamental problems in systems and circuit-level neuroscience.

John Rickfords research and teaching are focused on sociolinguistics the relation between linguistic variation and change and social structure. He is especially interested in the relation between language and ethnicity, social class and style, language variation and change, pidgin and creole languages, African American Vernacular English, and the applications of linguistics to educational problems.

The academy is a private, nonprofit institution that was created in 1863 to advise the nation on issues related to science and technology. Scholars are elected in recognition of their outstanding contributions to research. This years election brings the total of active academy members to 2,461.

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Six faculty elected to National Academy of Sciences - Stanford Today - Stanford University News

Introduction to Gene Therapy Using Viral Vectors

Gene therapy can adapt to each person to treat a variety of illnesses including cancer, rare diseases, and to promote wound repair. Currently, adeno-associated vectors, lentivirus, and retrovirus have been successfully implemented accounting for 19 FDA approved gene therapy products.1 Nine patients infused with AAV5-hFVIII-SQ, an adeno-associated vector serotype 5 (AAV5) that delivers exogenous factor VIII, were cured of Hemophilia B.2 This novel gene delivery system effectively treats Hemophilia A by producing blood-clotting proteins leading to fewer bleeding issues and cured patients with Hemophilia B. However, AAV vectors are difficult to scale-up and have been associated with toxicity and inflammation limiting their use in gene therapy.3 Comparatively, the use of a lentiviral vector for gene transfer cured a young boy of sickle cell anemia.4 While retroviral transduction of COL7A1 cDNA cured dystrophic epidermolysis bullosa by restoring C7 synthesis encoded by OL7A1 cDNA without host integration.5 However, lentiviral and retroviral vectors have limitations such as a low cloning capacity and integration into the host genome creating the potential for insertional mutagenesis. Moreover, there are potential safety concerns for the development of replication-competent retroviruses.6 The high cost, low scalability and biosafety concerns associated with current viral vectors, outlined in Table 1, highlight the large potential use of baculoviruses in gene therapy. Baculoviruses provide a relatively safe, scalable, and cost-effective vector for gene therapy.7

Table 1 Viral Vector Comparison for Gene Therapy

Baculoviruses, naturally known to infect Lepidoptera, have been exploited for their recombinant protein expression since 1983, enabling the development of a diverse range of therapeutics.8 Baculovirus gene delivery systems enable site-specific delivery, mitigating adverse effects, and improving therapeutics.9 This easily modifiable gene therapy system may be the cost-effective and efficient backbone needed for gene therapy. Following genomic sequencing of the individual, baculoviruses can be used to deliver the deficient genes or promote a proper biological response. Baculovirus vectors have already been implemented in several successful studies including cancer treatment, vaccines and regenerative medicine demonstrating their potential.1012 The diverse applicable use of baculoviruses generates a promising future for personalized medicine and gene therapy. Here we review the mechanism of baculovirus gene therapy and focus on optimizing it for individual treatments.

There are several types of baculoviruses that possess a high specificity to their natural insect hosts such as arthropods and Lepidoptera. Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori MNPV (BmMNPV) strains, ranging from 80180 kbp, are the most extensively studied in gene therapy.13,14 During baculovirus transcription and replication there are three main phases termed early, late, and very late. The early phase commences upon attachment, injection of the viral genome, uncoating, viral gene expression, and finally halting host transcription. Host transcription factors recognize and transcribe early viral genes within 0.5 to 6 hours post-infection.15 The activation of these genes allows for DNA synthesis and late gene production which are mostly structural proteins.15 During the late phase, the nucleocapsid structural protein with gp64 is produced enabling horizontal infection.16 The nucleocapsid then interacts with the nuclear membrane and becomes enveloped. Finally, viral promoters, polyhedrin and p10, are transcribed and hyper-expressed.17 The polyhedron then crystalizes around ODV forming occlusion bodies that fill the nucleus and fibrillar structures.17 Meanwhile, viral proteins, chitinase and cathepsin, assist with host cuticle breakdown.18 This cycle continues until there are many occlusion bodies (OBs) causing the insect to liquefy and rupture. The OBs account for 30% of an infected larvaes dry weight, and 25% of the cell protein produced is polyhedral capsules.19,20 This large and natural amplification feature makes baculoviruses an attractive potential for gene therapy where large scale gene production is necessary. The potential exploitation of the baculovirus life cycle for gene therapy can be seen in Figure 1. Following insect cell replication, the baculovirus vectors can be purified from the culture supernatant using heparin affinity chromatography.21 Purification concentrates the extracted baculovirus by 500-fold with a 25% infectious particle recovery rate. This can be scaled-up in a closed-system suspension culture generating sufficient clinical-grade vector levels for gene therapy.21 Alternative methods of purification include size-exclusion chromatography, monolithic ion-exchange chromatography, ion-exchange membrane chromatography, high-speed batch centrifugation, sucrose gradient centrifugation, and tangential flow ultrafiltration.

Figure 1 Lifecycle of baculoviruses (BV) and exploitation for recombinant protein production. Steps 111, in black text, describe the continuous lifecycle of baculoviruses, from infecting an insect to mass production of viral proteins. The red test indicates steps that be modified to produce the gene or protein of interest for therapeutic applications. The figure was created with BioRender.

Upon the discovery that baculoviruses could transduce mammalian cells, their therapeutic potential has rapidly expanded.22 The viral genome has since been modified and manipulated to improve the transduction efficiency and ease of production. Correspondingly, several vector systems have been developed including BacMam, Bac-to-Bac, MultiBac, and derivatives of these AcMNPV transfer vectors.2325

For foreign genes to be expressed, the viral or mammalian promoter must be recognized. Viral promoters p10 and polyhedrin have been most commonly used to promote transcription due to their high expression activity.14,26 However, a mammalian promoter can also be used to drive heterogeneous gene expression following viral transduction, termed a BacMam.23 BacMams can support gene insertions up to 40 kb but have a transient expression of four days without a selection force. Some mammalian promoters used to initiate gene transcription include Rous-sarcoma virus long terminal repeats (RSV-LTR), cytomegalovirus (CMV), simian virus 40 (SV40), chicken beta-actin (CAG), hepatitis B virus (HBV), human a-fetoprotein/ubiquitin C promoter, and drosophila heat shock protein 70 (hsp70) promoter.27 Viral and mammalian promoters can be used in conjugation with genomic enhancers to promote transgene transcription. Specifically, the insertion of an additional homologous region 1 (hr1) into baculoviruses has been used to activate mammalian promoters and results in improved stability, overexpression of the transgene, and prolonged transgene expression.13 A dual expressing BacMam vector (BV-Dual-s1) has since been produced. This system fuses s1 glycoprotein of avian infectious bronchitis virus with AcMNPV gp64 glycoprotein displaying the S1-gp64 on the viral surface.28 Moreover, vesicular stomatitis virus G (VSVG) glycoprotein has been incorporated under p10 promoter control allowing for viral surface display, enhanced transduction, and prolonged expression.26 However, this system can induce a strong humoral and cell-mediated immunity. The BacMam system also led to the development of BacMaM derivatives such as pFastBac1 and pFastBacmam.29 Specifically, pFASTBacMam-1 is driven by an SV40 promoter and a neomycin resistance marker, which allows for stable cell line selection after BacMam transduction.29 Promoter selection facilitates transcription and permits more strict controls over transgene expression.

Recombinant baculoviruses (rBVs) were first generated using homologous recombination in insect cells. This led to the development of the Bacmid system which uses bacterial artificial chromosomes containing E. coli fertility factor replicon maintained as a circular supercoiled extrachromosomal single-copy plasmid.23 The Bacmid system can accept 300 Kb gene inserts and can be modified using site-specific recombination.23 Homologous recombination can also delete background parental genes while repairing an essential gene like the orf1629 gene, essential for viral replication, or p10 genes allowing for purification.30,31 However, this technique only has a 1% transduction efficiency.32 This led to the development of flashBAC.33 The flashBAC method contains a partially deleted orf1629 gene so that homologous recombination can restore orf1629s function while eliminating bacterial sequences.33 Only rBVs have a functional orf1629 gene and can replicate allowing for easier purification. Other baculovirus genes have also been eliminated to improve foreign protein quality and yield.

New methods using primarily transposition also improved transduction efficiency. One of the first and most used systems is the Bac-to-Bac system.30 This system consists of three antibiotic selection markers (ampicillin, kanamycin, and gentamycin) and an intermediary transfer plasmid to insert foreign genes via targeted transposition. Specifically, Tn7-mediated site-specific transposition in E. coli is used to direct cassette integration and expression producing recombinant baculoviruses.30 This is still the only system that generates 100% pure recombinant baculoviruses (rBVs) without further purification. A similar system, Bac-2-the-Future (B2F), was developed based upon this Tn7 transposition method.24 However, the gentamycin resistance marker was replaced with pDP1381 reducing the number of false positives and vector size.24 These baculovirus systems provide the bases for site-specific gene delivery, within personalized medicine, compared to the standard systemic administration of common drugs.

Baculovirus production can be enhanced in insect cells by altering the chromatin state and media supplements. A more relaxed chromatin state facilitates accessibility for more efficient transcription. Sodium butyrate, trichostatin A and valproic acid all induce histone acetylation promoting chromatin accessibility and transgene expression.29,34 Similarly, histone deacetylation inhibitors induce histone hyperacetylation, relaxing the chromatin structure, and improving gene transcription and delivery.35 Media supplements also affect baculovirus transgene expression. Monteiro et al demonstrated that the addition of cholesterol to the media results in a 2.5-fold increase in baculovirus production and a 6-fold increase in virus-like particle (VLP) production.36 Similarly, the addition of glutathione, antioxidants, and polyamines resulted in a 3-fold increase in baculovirus production.36 These simple yet effective modifications can significantly enhance the efficiency and feasibility of baculovirus production for gene therapy.

A large advantage to BEVS is that they naturally generate proteins with proper phosphorylation and post-translational modification.37 Human-like glycosylation can also easily be achieved through genetic engineering enabling efficient treatment between individuals.38 Specifically, the N-terminal signal peptides are essential for directing the protein destination and fate. Native baculovirus signal peptides can be replaced by insect proteins like honeybee melittin or baculovirus proteins like gp64 to alter the protein fate.39,40 However, the difference in protein glycosylation between lepidopteran and higher eukaryotes can affect protein folding, degradation, location, and immunological response.38 N-glycosylation in insects also involves the transfer of preassembled oligosaccharide (Glucose3Mannose9N-acetylglucosamine2) from a lipid complex to an aspartate residue in the endoplasmic reticulum (ER) lumen.38 The protein then moves from the ER to the Golgi where enzymes trim and add sugar moieties to the glycan molecules. Comparatively, mammalian cells differ in that complex sugars with terminal sialic acids are added instead of sugar moieties. This led to the development of Sf9 and High five cells which encode bovine -1, 4-galactosyl transferase and rat -2, 6-sialyltransferase which enable proper addition of galactosyl and sialyl into proteins.37,41 Recently, Moremen et al developed an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases and other glycan-modifying enzymes to enable proper glycosylation disease and person-specific use.42

Other baculovirus modifications for optimal human use include gene deletions or insertions to prevent proteolytic cleavage or assist with protein folding. Specifically, genes such as chitinase and cathepsin, responsible for breaking down the insect cuticle, are not necessary for human therapeutic applications and can be replaced with genes of interest.31 Beneficially, the deletions of both of these bacculovirus genes results in increased levels of transgene proteins and ensures the transmission of viral occlusion bodies.18 Chaperone proteins often assist with protein modification, directing location and folding which corresponds to function. Cytosolic chaperones, like hsp70 and hsp40, prevent polypeptide aggregation and can be incorporated into the baculovirus genome to promote proper protein folding.43 Similarly, other chaperones such as binding immunoglobin protein, calnexin, calreticulin and protein disulfide isomerase can all assist with folding proteins produced from BEVS.44,45 A list of modifications that can enhance BEVS protein production, for therapeutic use, is outlined in Table 2.

Table 2 Enhancing Insect Cell Baculovirus Production

An essential step for gene delivery is the ability of the viral vector to enter the intended cell type. Advantageously, baculoviruses are capable of transducing both dividing and non-dividing cells. This includes common cell lines like HeLa, Huh-7, HepG2, bone marrow fibroblasts, PK1 cells, and human neural cells.8,46,47 However, transduction efficiency varies depending on cell type; 30% in undifferentiated human neural progenitor cells and 55% in differentiated cells.47 Specifically, gp-64 and heparan sulfate are required for mammalian cell entry.48,49 Several factors contribute to baculovirus production efficiency including cell type, chromatin state, promoter type, and protein expression. The ability of engineered baculoviruses to transduce specific mammalian cells reveals its potential for site-specific gene therapy and extension into personalized medicine.

Optimizing the virus method of cell entry and viral protein production is essential for therapeutic applications. Baculoviruses are capable of entering both permissive and nonpermissive cells, eliminating a common barrier to gene therapy.50 Specifically, the viral surface protein, gp64, is critical for efficient virus entry and endosomal escape in mammalian cells.51 The addition of another gp64 gene results in a 10 to 100-fold increase in reporter gene expression.39 Gp64 has also been fused to short peptide motifs of gp350/220 on Epstein-Barr virus (EBV) for enhanced gene delivery to B cells.52 Alternatively, co-expression of glycoproteins from thogotoviruses with gp64 improves virus-endosome fusion and endosomal escape resulting in a 4 to 12-fold increase in transduction efficiency.53 The high adaptability of baculoviruses elucidate its potential role in treating diseases in a person-specific manner.

The addition of several other molecules to the surface of baculoviruses has also enhanced transduction efficiency. Some of these additions into the baculovirus envelope include VSVG, influenza virus neuraminidase, single-chain antibody fragment, Spodoptera exigua MNPV (SeMNPV) F protein, endogenous retrovirus, and single antibody chains.26,5457 Specifically, Fc regions of antibodies enable antigen-presenting cells (APC) specificity.55 Similarly, the addition of VSVG demonstrated a 10 to 100-fold increase in transduction in human hepatoma and rat neuronal cells and broadened baculovirus tropism.58 VSVG has also been fused to tumor-homing peptides (LyP-1, F3, and CGKRK) on the baculovirus surface improving tumor binding 2-5-fold.59 Moreover, the strong attraction between avidin and biotin was exploited in avidin-displaying baculoviruses to increase transduction efficiency and correspondingly gene delivery.60 Chen et al fused a cytoplasmic transduction peptide to gp64 producing a cytoplasmic membrane penetrating baculovirus (vE-CTP).61 Simultaneously, the HIV Tat protein transduction domain was fused to the baculovirus capsid protein VP39 forming a nuclear membrane penetrating baculovirus (vE-PTD) improving transduction efficiency.61 Alternatively, cationic amino-functional poly (amidoamine) dendrimers complexed with baculoviruses enabled the binding of the cationic viral particles to the cell membrane.12 This strong interaction assisted with virus internalization and improved angiogenic vascular endothelial growth factor (VEGF) gene transfer and expression.12 Malaria proteins, three circumsporozoite protein variants and a thrombospondin-related anonymous protein, have also been added to the baculovirus envelope to enhance transduction efficiency in hepatocytes.62 Overall, the incorporation of diverse foreign proteins, into the baculovirus envelope, can be chosen to optimize transduction efficiency based on the disease and personalized needs.

As previously mentioned, the promoters used in baculovirus gene delivery systems can dictate transduction efficiency in gene therapy. The most commonly used viral promoters include polyhedron and p10. The fusion of heterologous genes at the 5 end of the gp64 gene, placed under the control of the polyhedrin or p10 promoter, allows viral envelope incorporation. Other viral promoters include p6.9, viral promoter 39, immediate early gene (IE1) promoter, and pB2, which have improved expression levels, particularly in early phases.63,64 Comparatively, in human mesenchymal cells, often the focus of regenerative medicine, human cytomegalovirus, ubiquitin C, phosphoglycerate kinase, and elongation factor-1 alpha (EF1) promoters have been incorporated into the Bac-to-Bac system.65 Particularly, EF1 demonstrated the highest transgene expression indicating the efficiency of the promoter is largely dependent upon cell type and more importantly revealing the potential for stem cell gene therapy. Moreover, promoters can be used in combination with transcriptional enhancers to increase transgene expression. For example, Gwak et al generated a baculovirus expression system with p6.9 promoter and transcriptional enhancers, homologous region 3 and repeated burst sequences, resulting in a 94-fold increase in foreign gene expression.66 Moreover, the stage of promoter expression can also alter gene expression. A 20-fold increase in transgene expression can be achieved using a very late promoter compared to an early promoter, in Drosophila melanogaster.50 The numerous combinations of viral and mammalian promoters enable adaptability and customization within baculovirus gene delivery.

rBVs have a relatively short transgene expression window of 714 days which can be optimized or extended based on the disease.67 Specifically, baculoviruses activate both the classical and alternative complement pathway leading to viral degradation and transient gene expression.68 Several methods have been employed to prevent complement activation and prolong gene expression. Activation of the alternative and classic complement pathway can be prevented through the display of decay-accelerating factor (DAF), factor H-like protein-1, C4b-binding protein, and membrane cofactor protein on the baculovirus envelope.69,70 Another study concluded that fusion of cluster of differentiation 46 and 59 with DAF (CD46-DAF-CD59) provides complement protection in HepG2 cells.71 Alternative envelope displays include VSVG, complement antibody C5, cobra venom factor, soluble, complement inhibitor I, compstatin and complement regulatory proteins.26,51,68 Moreover, Liu et al recently demonstrated that the BmNPV vector is more stable in human serum than AcMNPV.72 Hindering complement activation, through the above-mentioned methods, can effectively prolong gene expression and dampen the associated immune response for personalized approaches. Alternatively, the short baculovirus gene expression can be optimized for wound repair whereas genetically prolonged gene expression can be beneficial in anticancer therapy.

The addition of proteins onto the baculovirus envelope can be optimized for each individual and therapeutic use. Specifically, the insertion of VSVG extended gene expression to 178 days in DBA/2J mice and 35 days in BALC/c mice.26 Moreover, the incorporation of vankaryin (an anti-apoptotic gene) into a baculovirus vector increased cell viability and length of protein production.73 Similarly, BV-AAV hybrids have shown promise whereby gene expression lasted 90 days in rat brains.74 Similarly, Luo et al constructed a baculovirus with inverted terminal repeats (ITRs), the origin of plasmid replication (oriP)/EBV-expressed nuclear antigen 1 (EBNA1) and Sleeping Beauty (SB) transposon.75 They found that the SB system enabled gene expression for 77 days without antibiotic selection.75 Moreover, the incorporation and expression of an antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) inhibited prostate and human ovarian xenograft tumor growth.75 More recently, Wang et al generated a bivalent hybrid baculovirus that displayed DAF and eGFP mediated by SB transposon system which prolonged the expression of hEA genes to 90 days.76 Moreover, the hEA genes exhibited antitumor effects in hepatocellular carcinoma xenograft mouse models as well as complement resistance.76 Alternatively, two baculovirus vectors have been used to generate a self-replicative episome providing constant gene expression for 48 days.77 Here, one vector encoding flippase recombinase cleaves and activates the other encoding oriP/EBNA1 from EBV and gene of interest within the Frt flanking region.77 Alternatively, viral components can be combined with non-viral such as fibrin gels to further prevent bleeding and promote wound healing. Previously, fibrin gels and BacMam-mediated gene delivery modulated gene release, enhanced transduction efficiency and prolonged gene expression in vivo.78 Methods of baculovirus optimization for gene therapy are described in Table 3, below.

Table 3 Optimizing Baculoviruses in Mammalian Cells for Gene Therapy

With the basis of BEVS established, more systems worked on improving protein quality and yield for therapeutics. Top-Bac was able to increase protein yield by 300%.80 Top-Bac uses several promoters some of which are hybrid sequences formed from late and very late AcMNPV genes. Moreover, Steele et al were able to generate a cell line with vankryin directly incorporated improving yield.73 Several other studies have looked into the genetic makeup of baculoviruses to better understand which genes can be manipulated or even removed. It was found that the combination of PCR and transformation-associated recombination, in yeast, generated a synthetic baculovirus genome based upon AcMNPV (AcMNPV-WIV-Syn1).81 The synthetic baculovirus omitted baculovirus genes enhancing recombinant protein production.81

Another barrier to viral gene therapy is the complexity and cooperation of native proteins. Beneficially, the large cloning capacity of BEVS allows for the production of several proteins or complex structures like virus-like particles (VLPs). Berger et al incorporated an array of small synthetic DNA plasmids termed acceptors and donors.25 The acceptors can be loaded with several genes to produce eukaryotic protein complexes with many subunits, termed MultiBac.25 This system enabled the discovery, understanding and treatment of complex molecules which was previously inaccessible. Similarly, Weissmann et al were able to assemble a rBV producing 25 individual genes in just 6 days.82 This method uses Gibson assembly reaction along with concepts from MultiBac earning the name biGBac.82 Comparatively, Zhang et al used a Uracil-specific Excision Reagent ligation-free cloning method.28 This enabled the targeted expression of multi-subunit anaphase-promoting complex within MultiBac, under the polyhedrin or chitinase gene loci, producing 13 proteins.28 The expression of multi-complex or multi-subunit proteins is essential for proper protein function and can be tailored to each individuals treatment providing a functional pathway, not just a protein.

Advantageously, the large cloning capacity of baculoviruses allows for large gene insertions (proteins, viral particles and more). The prolonged gene expression of AAV vectors can be combined in BEVS to prolong transgene expression. The first recombinant AAV (rAAV) treatment, derived from baculoviruses, successfully treated familial lipoprotein lipase deficiency (LPLD), Glybera.83 Although successful, the large $1-million cost led to the treatments withdrawal from the market. OneBac appears to be a more affordable option by using a stable insect Sf9 cell line with silent copies of inducible AAV1012 Rep and Cap genes.84 The combination of AAV vectors with OneBac increases the yield of genomic particles and functional particles by 6-fold and 20-fold, respectively.85 Similar beneficial results were seen in hypopharyngeal carcinoma gene therapy where Bac-Adeno-Associated viral vectors with Luc-P2A-eGFP or sodium iodide symporter (NIS), under CMV promoter control, infected bone marrow mesenchymal cells (BMSCs).86 The BMSCs effectively took up radioactive iodine demonstrating its potential to act as a targeted-delivery vehicle in mice.86 More recently, Wu et al developed a new combination vector using ribosome leaky-scanning to express AAV Rep and Cap proteins downstream polh and p10 promoters, respectively.87 The rAAV genome can be inserted between two Bac promoters yielding 105 vector rAAV2/8/9 genomes from Sf9 baculovirus-infected cells.87 This indicated that BEVS may be suitable for large-scale rAAV production as well as targeted cell therapy. This is particularly useful in treating diseases like cancer with high heterogeneity.

Baculoviruses can also be exploited within vaccines and treatments for immune diseases through immunological modifications. Cytoplasmic sensors like retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-association protein 5 (MDA5) recognize dsRNA activating the interferon-beta promoter stimulator (IPS-1) mediated signal pathway resulting in interferon type 1 (IFN-1) production.34 This is accompanied by activation of toll-like receptors 3/7/9 which are endosomal sensors that recognize viral DNA, RNA and intermediate RNA, respectively.34 This leads to the activation of IRF3/7 and NF-k (nuclear factor kappa light chain enhancer of activated B cells) in macrophages and dendritic cells.34 Ultimately thisleads to the production of IFN-1, inflammatory cytokines, and inflammatory chemokines, all of which promote inflammation, and viral DNA degradation. This immune activation can be exploited in vaccine candidates providing a safe, personalized and scalable vector.

Moreover, the incorporation of foreign proteins into the baculovirus envelope or nucleocapsid core can be used in gene therapy. Baculovirus proteins expressed on the viral surface or nucleocapsid core can elicit a humoral immune response or activate MHC I leading to activation of CD8+ T cells, respectively.88,89 Baculovirus surface peptide display demonstrated a strong adjuvant activity protecting against lethal viruses like influenza and encephalomyocarditis.34,90 Influenza immunity has been induced by Hemagglutinin (HA) expression on baculovirus using Bmg64HA HA fragment of H5N1 fused to the gp64 gene.91 Alternatively, baculoviruses can be used for VLP production like in severe acute respiratory syndrome (SARS), human immunodeficiency virus (HIV), Sudan virus, Ebola virus, Marburg virus, rabbit hemorrhagic disease virus (RHDV) and Rous sarcoma virus.9297 More recently, Hinke successfully constructed a BEVS with a recombinant 65 kDa glutamate decarboxylate, Diamyd, to treat type 1 diabetes.98 Evidently, BEVS surface display and VLP production can be customized for personalized vaccines and treating heterogeneous diseases.

The display of surface proteins can also direct cell-specific uptake of baculoviruses. Currently, Fc receptors, folate, and epidermal growth factor (EGF) have been used to dictate baculovirus selectivity.99 Rty et al exploited the avidin-biotin interaction to increase transduction efficiency while expressing biotinylated EGF causing the system to target EGF displaying cells.60 Polyethylene glycol (PEG)-folate has also been displayed on the baculovirus surface to target the Fc receptors displayed specifically on malignant cells enabling targeted gene delivery.100 In comparison, rBVs displaying human epidermal growth factor-2 (HER2) single-chain variable domain fragments (scFV) while expressing Apoptin bind specifically HER2 positive SK-BR-3 breast cancer cells reducing cancer cell viability.101 Similarly, a rBV expressing BIMs, a strong apoptosis inducer, resultedin selective death of HCV-positive cells only further proving BVs potential for selective gene therapy.102 The selective treatment of an individuals malfunctioning or impaired cells can mitigate the systemic and adverse effects seen in traditional medical treatments, significantly improving the quality of treatment, care, and life. Consequently, baculovirusescan be exploited in regenerative medicine (Table 4), anti-cancer treatments (Table 5), and vaccine vectors.

Table 4 Baculoviruses in Therapeutics and Regenerative Medicine

Table 5 Baculoviruses in Cancer Treatment

The large cloning capacity of baculoviruses enables transgene expression of large multi-complex proteins both in vivo and ex vivo. This is particularly useful for use in anticancer therapy, stem cell regeneration and in vaccine development. Specifically, a toxin vector for diphtheria toxin A has been developed to eliminate malignant glioma cells within the brain.106 Other rBVs expressing normal epithelial cell specific-1 and herpes simplex virus-1 thymidine kinase have shown similar promising results in eliminating glioblastoma and gastric cancer cells.107,108 Moreover, angiogenesis-dependent tumours have been treated with a hybrid SB-Baculovirus vector to prolong antiangiogenic fusion protein expression (endostatin and angiostatin).75 Lin et al engineered bone marrow-derived mesenchymal cells (BMSCs) to express bone morphogenetic protein 2 and VEGF enabling enhanced femoral bone repair and bone quality.109 Similarly, for myocardial infarction therapy, baculoviruses can be engineered to expressed Angiopoietin-1 to increase capillary density,reduce infarct sizes and other clinically fevaourable conditions in experimental rats.110

rBVs also have a large potential in VLP and vaccine production. One of the first vaccines using baculoviruses, called FluBlok, used the HA antigen as a subunit vaccine to elicit a protective immune response.29 This technique has been extended into other vaccines such as human papillomavirus, prostate cancer and familial lipoprotein lipase deficiency.10,111,112 The three vaccines expressed HPV-L1 protein, granulocyte macrophage colony-stimulating factor and an AAV vector with lipoprotein lipase transgene, respectively. Moreover, the administration of baculoviruses was capable of eliminating malaria parasite in mice liver and eliciting a protective humoral and cellular immune response.113 The scalability of BEVS are beneficial for mass production of molecules like VLPs. It is predicted that baculoviruses are capable of generating 415 million 10 g/dose vials of anti-flu vaccines in one week compared to the 6 months standard using chicken embryos.114 The high protein production and efficacy supports the use of baculoviruses as a promising vaccine vector and scalable approach to personalized medicine. Current vaccines involving baculoviruses are included in Table 6, below.

Table 6 Baculoviruses in VLP Production and Vaccines

There are a few limitations associated with baculovirus in gene therapy, hindering its wide-scale use and production. Specifically, BEVS can induce an immune response producing inflammatory cytokines and chemokines and activating the complement pathway. This can lead to an unnecessary immune response and viral genome degradation if used for non-vaccination purposes. Upon serum contact baculoviruses activate RIG-I/IPS-1 or cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway which can suppress transgene expression.130 Moreover, baculoviruses exhibit transient gene expression. Without selection, gene expression typically lasts 714 days in most cell lines, including CHO, HeLa and BHK.67 However, several gene insertions or modifications have been able to extend gene expression and prevent complement recognition.75,77,131 Transgene expression can also be prolonged by shielding the baculovirus from the immune system using a polymer coating. This prevents immune activation and prolongs gene expression and its associated therapeutic effect. Alternatively, the transient gene expression mitigates safety concerns providing potential in vaccine vector or adjuvant field. Another limitation of baculovirus vector systems is the virus fragility. The half-life of the virus is only 173 hours at 27C and 78 hours at 37C.44 Moreover, defective interfering (DI) particles accumulate during serial cell culture passages. The amount of DI particles can be reduced by using a low MOI or by removing the non-hr origin from the SeMNPV baculovirus genome preventing DI formation for 20 cell passages.132

Future outlooks of baculoviruses in therapeutics are exciting and very promising. This potential has been recently recognized worldwide such as in project Baculogene. This project focuses on developing methods for large-scale production, downstream processing, purification and analysis methods for direct baculovirus applications in gene therapy. More recently, baculoviruses have been used in four pre-clinical COVID-19 vaccines, highlighting its use and adaptability. Specifically, baculoviruses were used to produce viral S protein and receptor binding domain protein in three subunit vaccine candidates as well as for VLP production in the fourth vaccine.133 The ease of genetic manipulations to extend transgene expression, prevent complement recognition, improve transduction efficiency, increase protein yield, and include several proteins at once, promote the feasibility and implementation of personalized medicine. This simple yet cost-effective scale-up method can be used to produce the exact dose and customized based on the genetic information of each individual.

Baculoviruses have excellent therapeutic potential in a number of diseases. They have been sucessfully used in vaccine industry, anticancer therapy, and recombinant protein productions. Their associated limitations may be quickly overcome through further genetic engineering and other methods. Moreover, the relative ease of production, non-replicative nature in mammalian cells, large gene(s) pay load, stability of the genes, advanced delivery features, and other methods continue to make them ideal for gene therapy, personalized medicine and other applications. Baculoviruses have a large potential to be optimized for each disease and individual through targeted gene and dose modifications. The simple production, protein extraction, and easy manipulation of insect cells provide the cost-effective method needed to advance gene therapy and personalized medicine.

This work is supported by the Canadian Institute of Health Research (CIHR) (grant # 252743). The figure was created using biorender.com

The authors report no conflicts of interest.

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A review of baculovirus vectors in gene therapy | BTT - Dove Medical Press

Fourteen Yale faculty members who work across a range of disciplines were among the 252 accomplished individuals elected to the American Academy of Arts & Sciences last week.

Those elected are extraordinary people who help solve the worlds most urgent challenges, create meaning through art, and contribute to the common good, said the academy in announcing the new members, who include artists, scholars, scientists, and leaders in the public, nonprofit, and private sectors.

We are honoring the excellence of these individuals, celebrating what they have achieved so far, and imagining what they will continue to accomplish, said David Oxtoby, president of the American Academy of Arts & Sciences. This past year has been replete with evidence of how things can get worse; this is an opportunity to illuminate the importance of art, ideas, knowledge, and leadership that can make a better world.

The academy was founded in 1780 by John Adams, John Hancock, and others who believed the new republic should honor exceptionally accomplished individuals and engage them in advancing the public good.

The new members from Yale are:

Dirk Bergemann, the Douglass and Marion Campbell Professor of Economics and professor of computer science, whose research is focused on game theory, contract theory, venture capital, and market design. He has made important contributions to the theory of mechanism design and has pioneered work on consumer behavior and dynamic pricing structures.

Ronald Breaker, Sterling Professor of Molecular, Cellular, and Developmental Biology and professor of molecular biophysics and biochemistry, who conducts research on the advanced functions of nucleic acids, including ribozyme reaction mechanisms, molecular switch technology, next-generation biosensors, and catalytic DNA engineering. His lab established the first proofs that metabolites are directly bound by messenger RNA elements called riboswitches, among other important discoveries.

Nancy Brown, the Jean and David W. Wallace Dean of the Yale School of Medicine and C.N.H. Long Professor of Internal Medicine, who is committed to medical education and mentorship.

Her own research has defined the molecular mechanisms through which commonly prescribed blood pressure and diabetes drugs affect the risk of cardiovascular and kidney disease, and in her clinical practice, she has treated patients with resistant and secondary forms of hypertension.

Hui Cao, the John C. Malone Professor of Applied Physics, whose research focuses on understanding and controlling quantum optical processes in nanostructures. Her work involves nanofabrication, material characterization, optical measurement with high spatial, spectral, and temporal resolution, and numerical simulation.

BJ Casey, professor of psychology, who is considered a world leader in human neuroimaging and its use in typical and atypical development. She uses brain imaging to examine developmental transitions across the life span, especially during adolescence. She heads the Fundamentals of Adolescent Brain Lab, and is a member of the Justice Collaboratory at the Yale Law School and the Interdepartmental Neuroscience Program.

Valerie Hansen, the Stanley Woodward Professor of History, whose scholarly expertise is on China before 1600, Chinese religious and legal history, and the history of the Silk Road. She most recently authored The Year 1000: When Explorers Connected the World and Globalization Began.

Arthur L. Horwich, Sterling Professor of Genetics and professor of pediatrics, a pioneer in the field of molecular chaperones and their role in protein folding in the cell and in neurodegeneration. His discoveries have advanced an understanding of the relevance of protein misfolding in diseases such as Alzheimers.

Gregory Huber, the Forst Family Professor of Political Science and chair of the political science department, who studies American politics and political economy. He is interested in understanding how interactions among the mass public and elites, political institutions, and policies explain important outcomes.

Akiko Iwasaki, the Waldemar Von Zedtwitz Professor Immunobiology and Molecular, Cellular, and Developmental Biology, and professor of epidemiology (infectious diseases), whose research focuses on the mechanisms of immune defense against viruses at the mucosal surfaces. Most recently, she has advanced understanding of SARS-CoV-2 and virus mutations.

Marcia K. Johnson, Sterling Professor Emeritus of Psychology, whose work has focused on memory and cognition, especially how complex memories are created, memory disorders, and the relation between emotion and cognition. She directs the Memory and Cognition Lab at Yale, which also studies cognition changes associated with aging.

Frederick J. Sigworth, professor of cellular and molecular physiology and biomedical engineering and of molecular biophysics and biochemistry, whose research centers on the structure and function of ion channels, which are central to many physiological processes. His laboratory is developing new computational and experimental methods for imaging membrane proteins in membranes.

Daniel A. Spielman, Sterling Professor of Computer Science and professor of statistics and data science and of mathematics, whose broad research interests include the development of fast algorithms for large computational problems often found in machine learning, scientific computing, and optimization. He was awarded a MacArthur Fellowship for this work and most recently won the Held Prize for helping solve a theoretical problem that mathematicians had been working on for decades.

Kathryn Tanner, the Frederick Marquand Professor of Systematic Theology, whose research relates the history of Christian thought to contemporary issues of theological concern using social, cultural, and feminist theory. One of her contributions was to illuminate the role that Christian faith and practice can have on the global economic system.

Ebonya L. Washington, the Samuel C. Park Jr. Professor of Economics, who specializes in public finance and political economy with research interests in the interplay of race, gender, and political representation. She also studies behavioral motivations and consequences of political participation and the processes through which low-income Americans meet their financial needs.

Joining the Yale faculty members as new members are such noted individuals as neurosurgeon and CNN medical correspondent Sanjay Gupta; playwright, screenwriter, and actor Suzan-Lori Parks; songwriter and performer Robbie Robertson; atmospheric scientist Anne Thompson; and media entrepreneur and philanthropist Oprah Winfrey. Benjamin Franklin was elected a member in 1781, and since then other honorees have included Alexander Hamilton, Ralph Waldo Emerson, Charles Darwin, Margaret Mead, Martin Luther King Jr., Anthony Fauci, Antonin Scalia, and Anna Deavere Smith.

The list of all new members is available on the academys website.

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Fourteen Yale faculty elected to American Academy of Arts & Sciences - Yale News

Crusoe has reduced flaring by over 1 billion cubic feet since inception and has the potential to reduce greenhouse emissions by the equivalent of hundreds of thousands of cars

Crusoe Energy Systems Inc. (the "Company") has closed a $128 million Series B equity financing led by Valor Equity Partners with participation from Lowercarbon Capital, DRW Venture Capital, Founders Fund, Bain Capital Ventures, Coinbase Ventures, Polychain Capital, KCK Group, Upper90, Winklevoss Capital, Exor, Zigg Capital and JB Straubel, the co-founder and former CTO of Tesla and founder and CEO of Redwood Materials. Crusoe also secured a non-dilutive $40m project financing facility from Upper90 in addition to the new equity capital. The combined funding will expand Crusoes operations as the Company pursues its mission to eliminate the routine flaring of natural gas and associated methane emissions while delivering low cost computing infrastructure. Crusoe deploys mobile, modular data centers that generate electrical power from otherwise wasted and flared natural gas (Digital Flare Mitigation or DFM).

Highlights:

Crusoe raised $128 million from leading technology and climate-focused investors

Fundraising follows Crusoes successful deployment and operation of 40 flare-powered data centers with oil producers in four states

Existing energy clients include leading operators with ambitious environmental targets such as Devon Energy, Kraken Oil & Gas, Enerplus and others; Crusoe has also previously operated DFM technology for Equinor, Norways state energy company and a leader in environmental excellence

Early cloud computing users include Massachusetts Institute of Technologys Computer Science and Artificial Intelligence Lab (MIT-CSAIL), Folding@Home (a COVID-19 therapy research consortium) and OpenCV (a leader in computer vision technology)

Crusoe aims to expand to more than 100 units over the coming year

Each Crusoe Digital Flare Mitigation system reduces CO2-equivalent emissions by up to 8,000 tons per year, equivalent to taking about 1,700 cars off the road

Natural gas flaring and methane emissions are increasingly targeted by investors, activists and regulators as a low-hanging opportunity to achieve climate goals

Crusoe currently operates 40 modular data centers powered by otherwise wasted and flared natural gas. Crusoes patented Digital Flare Mitigation technology has been deployed in North Dakota, Montana, Wyoming and Colorado. The Company plans to grow to more than 100 units over the next year as it expands within new and existing flaring-intensive markets as well as locations with oversupplied wind or solar power. Since launching in 2018, Crusoe has emerged as a scalable solution to reduce flaring through energy intensive computing such as bitcoin mining, graphical rendering, artificial intelligence model training and even protein folding simulations for COVID-19 therapeutic research.

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"We welcome Valor as our new lead investor along with climate-focused investors like Lowercarbon Capital that align with Crusoes mission to eliminate routine flaring in the oilfield," said Chase Lochmiller, the CEO and co-founder of Crusoe. "Valor brings tremendous expertise in scaling technically and operationally complex businesses as illustrated by their success partnering with the management teams at Tesla, SpaceX and others."

"Crusoe provides the type of cross-cutting solution that solves multiple technological, energy, and climate challenges simultaneously," said Antonio Gracias, Valor founder, CEO and CIO. "The financing announced today will help to scale Crusoe by orders of magnitude, meaning we can unlock vast and economic computing resources for technology users while eliminating significant climate-harming emissions." Valor has been focused on sustainability and climate change for well over a decade with investments like Tesla, SolarCity, Misfits Market, AMP Robotics and more. In addition, Valor has been an early investor in crypto infrastructure technology through businesses like BitGo and others. "Our investment in Crusoe builds on our track record of supporting world-class entrepreneurs in building great companies using cutting-edge technology to improve the world."

Crusoes solution arrives amid escalating efforts by industry, regulators and financiers to rapidly reduce flaring and methane emissions:

New Mexico recently passed new laws limiting flaring and venting to no more than 2% of an operators production by April of 2022.

North Dakotas legislature has voted in favor of new incentives aimed at supporting on-site flare capture systems including Digital Flare Mitigation, a measure that has attracted bipartisan support in the state.

Wyomings governor recently signed House Bill 189 into law, which creates incentives for the reduction of gas flaring through cryptocurrency mining

BlackRocks management called for a complete end to routine flaring by 2025 in a recent letter to investors.

The World Bank has launched a "Zero Routine Flaring by 2030" initiative with endorsement from 34 governments and 44 oil companies.

The Environmental Defense Fund recently published a broad survey of flaring, which indicates that 3.5 times more methane escapes from flares than previously estimated by the EPA.

Numerous leading oil companies have published environmental goals aimed at steep reductions in both flaring and methane emissions.

By displacing loads from the grid and preventing the methane leakage associated with natural gas flaring, each Crusoe modular datacenter reduces CO2-equivalent emissions by up to 8,000 tons per year, equivalent to taking about 1,700 cars off the road. Methane is approximately 84 times more potent than CO2 as a greenhouse gas, so by preventing methane leakage from flaring, Crusoes technology reduces CO2-equivalent emissions by up to 63% relative to continued flaring.

"Crusoe is a mission-driven company," said Cully Cavness, Crusoes co-founder, president and chief operating officer. "Our team is unified around the goal of solving the environmental challenges of stranded energy, especially flare gas. This means working with industries that have a large environmental impact to help clean them up. At Crusoe we understand that environmental solutions scale best when they are economic. Digital Flare Mitigation offers exactly that - a scalable economic solution to a major environmental problem."

About Crusoe Energy Systems Inc.

Crusoe Energy Systems provides innovative solutions for the energy industry. By converting natural gas to energy-intensive computing, Crusoes Digital Flare Mitigation service delivers an environmentally sound way to create a beneficial use for otherwise wasted natural gas. Crusoe has deployed flare mitigation projects in Wyomings Powder River Basin oilfield, Colorados Denver-Julesburg oilfield and North Dakota and Montanas Bakken oilfield. Systems are scalable up to millions of cubic feet per day and can be deployed rapidly to even the most remote locations.

Please reach out to info@crusoeenergy.com or visit http://www.crusoeenergy.com to learn more, and follow Crusoe on Linkedin and Twitter.

View source version on businesswire.com: https://www.businesswire.com/news/home/20210426005202/en/

Contacts

Crusoe Energy Systems:Cully Cavness, info@crusoeenergy.com

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Crusoe Achieves Operational Milestones and Closes $128 Million Series B Financing to Expand Patented Digital Flare Mitigation Technology - Yahoo...

Abstract

The delivery of therapeutics through the circulatory system is one of the least arduous and less invasive interventions; however, this approach is hampered by low vascular density or permeability. In this study, by exploiting the ability of monocytes to actively penetrate into diseased sites, we designed aptamer-based lipid nanovectors that actively bind onto the surface of monocytes and are released upon reaching the diseased sites. Our method was thoroughly assessed through treating two of the top causes of death in the world, cardiac ischemia-reperfusion injury and pancreatic ductal adenocarcinoma with or without liver metastasis, and showed a significant increase in survival and healing with no toxicity to the liver and kidneys in either case, indicating the success and ubiquity of our platform. We believe that this system provides a new therapeutic method, which can potentially be adapted to treat a myriad of diseases that involve monocyte recruitment in their pathophysiology.

Hypovascularity in pancreatic ductal adenocarcinoma (PDAC) (1) and reduced blood supply to the heart following ischemic myocardial injury mean that sole reliance on drug delivery through the circulatory system is ineffective under these conditions; therefore, if this method is to be used to achieve efficient delivery of drugs to target locations, then augmentation will be needed. Vascular permeability has been used as a method of passive drug delivery (2); however, studies have shown that this phenomenon occurs only transiently in the heart and the available time window is not long enough for meaningful delivery of therapeutics (3, 4). This makes low vascular permeability a bottleneck that greatly hampers drug efficacy and deliverability. Therefore, a drug delivery platform capable of leaving the circulatory system, regardless of vascular permeability, and infiltrating deep into the disease site is attractive.

Recruitment of immune cells, such as monocytes, takes place as a natural response to a change in the physiological environment. The role of monocytes varies. In the tumor microenvironment, as a cancer-related inflammatory response, they are constantly recruited and are capable of infiltrating into the tumor site (5, 6), while after myocardial injury, splenic monocytes are recruited and are capable of infiltrating into the heart to help heal the myocardium (7, 8). Inspired by this phenomenon, we designed a lipid nanoparticle (LNP)based drug delivery platform with an active targeting scaffold that acts as a vehicle and is capable of selectively attaching onto the surface of circulating monocytes in the blood stream, moving with them, and extravasating together with them into the diseased site.

The body consists of a myriad type of cells, and targeting a specific cell type is therefore challenging. One possible way to achieve this is to use a cell-specific ligand as the targeting scaffold. As an example, several studies have reported nanoparticles carrying macrophage-specific ligands in their cargo as therapeutics. These nanoparticles were able to deliver the ligands into the macrophages resulting in their activation (9). Although this kind of ligands can potentially be used as a targeting scaffold, we chose not to use them, as we only aim to attach our nanoparticles on the monocyte surface without activating them. Furthermore, some of the ligands may not be monocyte specific and may also target endothelial cells (10, 11), resulting in unwanted off-target accumulation. Taking these into consideration, we avoided using ligands as the targeting scaffold and we opted to use aptamers instead.

Aptamers are synthetic short, single-stranded DNA or RNA oligonucleotides used as biotechnological tools and therapeutic agents. They can be designed to have high affinities toward specific proteins through their folding into tertiary structures (12). The idea of using oligonucleotides to target proteins emerged in the early 1990s, and since then, aptamers have been widely applied in many fields, including food safety, environmental monitoring, clinical diagnosis, and therapy (12). With the development of cell systematic evolution of ligands by exponential enrichment (Cell-SELEX), it has become possible to design and select aptamers with high affinities toward specific cells types, such as monocytes, while avoiding unwanted bindings to endothelial cells (13). In this study, we took advantage of this advanced technique to select a specific monocyte-targeting aptamer and integrated it with our LNP as an active-targeting scaffold to produce a high-affinity monocyte-targeting drug delivery vehicle.

Several studies have described a similar strategy whereby the bodys own cells were used to carry nanoparticles to diseased sites. T cells carrying nanoparticles loaded with a topoisomerase inhibitor ligand SN-38 were reported to reduce tumor burden in mice with disseminated lymphoma (14). LNPs carrying tumor necrosis factorrelated apoptosis-inducing ligand were able to attach onto the surface of leukocytes and kill colorectal and prostate cancer cells, as well as circulating tumor cells in mice (15). Furthermore, by hitchhiking on the surface of red blood cells, nanogels carrying reteplase, a thrombolytic enzyme, ameliorated pulmonary embolism in mice (16). Our strategy, on the other hand, makes use of monocyte recruitment to the diseased site. We hypothesize that because the recruitment is an active process, it ensures that the nanoparticle and its cargo can reach the site it is intended. We also hypothesize that our monocyte-targeting drug delivery platform is versatile and can be used to treat myocardial ischemia-reperfusion (IR) injury and pancreatic cancer, two very different deadly diseases, which involve the monocyte recruitment phenomena that we harness in our strategy.

IOX2, a potent and selective hypoxia-inducible factor (HIF)1 prolyl hydroxylase2 inhibitor, is capable of preventing proteasome-mediated degradation of HIF-1 (17, 18). The HIF-1 protective effect of IOX2 not only contributes to the reduction of apoptosis but also enhances the transcription responses of HIF-1 (19, 20). Gemcitabine is a common chemotherapeutic agent for pancreatic cancer. It is a deoxycytidine analog capable of inhibiting the DNA replication in cancer cells and causing cell death (21). We encapsulated both of these drugs separately into our delivery vehicle, and by doing so, we were able to successfully ameliorate IR injury (using IOX2-loaded nanoparticles) and reduce tumor burden in PDAC mice (using gemcitabine-loaded nanoparticles). Moreover, unlike other bio-based materials, our aptamer-based scaffold is not patient specific, synthetic, and can be chemically modified, which are highly advantageous traits in the clinical setting.

As our delivery of therapeutics to disease sites relies on the recruitment of monocytes, we first examined the most efficient time point for delivery by constructing monocyte recruitment profiles to the injured heart and tumor site using IR (Fig. 1, A to C) and PDAC (Fig. 1, D to F) models of transgenic CCR2RFP/+ mice, respectively. We observed an increase in the number of recruited monocytes following IR injury and PDAC model establishments, which reached a maximum at day 4 after IR injury (Fig. 1B) and day 7 after KPC (KrasG12D, p53fl/fl, Pdx1-Cre) tumor cell transplantation (Fig. 1E). Furthermore, the number of circulating monocytes after IR injury and KPC tumor cell transplantation showed significant difference until 5 hours and day 14, respectively (figs. S1 and S2). Recruitment of monocytes to the IR heart was further confirmed by fluorescence-based intravital microscopy of the heart, whereby CCR2RFP/+ monocytes were observed (Fig. 1C). In the PDAC model, transplantation success and recruitment of monocytes were further confirmed by fluorescence-based intravital microscopy, whereby green fluorescent protein (GFP)+ KPC cells and red fluorescent protein (RFP)+ CCR2 monocytes were clearly observed at the injection site (Fig. 1F).

(A) The in vivo imaging system (IVIS) revealed CCR2RFP/+ cell recruitment to the injured heart after IR. (B) IVIS quantification of the CCR2RFP/+ recruitment to the injured heart after IR. (C) Recruitment of CCR2RFP/+ cells in the injured heart after IR under an intravital microscope. (D) Representative IVIS images of CCR2RFP/+ monocyte recruitment in a mouse orthotopic pancreatic cancer (PDAC) model. The mouse KPC cells were luciferase and GFP double transgenic. (E) IVIS quantification of CCR2RFP/+ monocyte recruitment in the tumor site. (F) CCR2RFP/+ recruitment in the PDAC model under an intravital microscope. (G) Schematic illustration of the aptamer-based LNP delivery approach in the mouse cardiac IR and PDAC models via circulating monocytes. (H) Flow cytometric analysis of the specificity of J10 aptamer to monocyte cell lines RAW264.7 and J774A.1, as well as mouse endothelial cell line SVEC. The S2 aptamer was a random ordering of the J10 aptamer sequence. (I) Flow cytometry showed ex vivo targeting of Cy5-labeled J10 aptamer against mouse monocytes. (J) In vivo targeting of J10 aptamerdecorated quantum dots QD655 to circulating CCR2RFP/+ and CX3CR1GFP/+ monocytes via intravital imaging. (K) Polymerase chain reaction (PCR) analysis of J10 aptamer accumulation in the infarct area after cardiac IR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. One-way analysis of variance (ANOVA) with a Tukey adjustment was used to analyze data in (B) and (I). Two-way ANOVA with a Tukey adjustment was used for data analysis in (E) and (H). Unpaired Students t test was used to analyze data in (K). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars, 100 m (C and F) and 20 m (K).

Aiming to produce a nanoplatform capable of binding to monocytes, we used nontoxic liposome-based nanoparticles coated with aptamers as a targeting scaffold, which are envisioned to be capable of infiltrating into the injured myocardium and pancreatic tumor site along with the monocyte (Fig. 1G). Aptamer candidates were chosen through the SELEX process against two monocyte/macrophage cell lines, RAW264.7 and J774A.1, for positive selection and the murine endothelial cell line, SVEC, for negative selection. Aptamers specific to both monocyte cell lines but not to SVEC were amplified through polymerase chain reaction (PCR). Following several rounds of SELEX, we identified aptamer J10 as the best candidate. The sequence of J10 was then scrambled to yield a control aptamer, S2 (fig. S3, A to E). The structures of both aptamers were predicted by Mfold software (22) (fig. S3, F and G). We then thoroughly investigated the capability of both aptamers to bind selectively to monocytes in vitro, in vivo, and ex vivo. Binding assays with Cy5-labeled aptamers confirmed that J10, but not S2, was capable of binding selectively to mouse monocyte cell lines (RAW264.7 and J774A.1) in vitro (Fig. 1H) and circulating myeloid (CD45+ CD11b+) cells ex vivo (Fig. 1I). Moreover, using intravital imaging to visualize the binding between circulating monocytes and QD655-labeled J10 (Fig. 1J and movies S1 to S4) clearly demonstrated that J10 selectively bound to monocytes. In vivo, intravenous injection of J10 and S2 aptamers revealed more J10 aptamer accumulated in the hearts with IR compared to S2 (Fig. 1K). J10 aptamer also has a higher binding affinity toward human monocyte cell lines THP-1 and U937, but not human endothelial cell line HUVEC, compared with S2 (fig. S4). All of these results supported our hypothesis that J10-labeled scaffold is capable of attaching selectively onto monocyte surface, which we then exploit to target the diseased sites.

After we successfully identified J10 as the candidate for monocyte-targeting drug delivery platform, we then endeavored to use it as an active-targeting scaffold on the nanoparticles for the treatment of IR injury. LNPs were synthesized using a thiolated linker DNA that can readily conjugate to maleimide-containing DSPE-PEG (1, 2-distearoyl-Sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]). The resulting DSPE-PEGlinker lipid was capable of hybridization with the aptamers (J10 or S2) to give the final monocyte-targeting LNP end product. Optimal aptamer density was determined through optimization of the molar ratio of linker:lipid, which was found to be 0.3%. A higher ratio, which translates to a higher density, did not result in a higher binding affinity to monocytes (fig. S5). Following self-assembly and encapsulation of the intended drugs (IOX2 or gemcitabine), aptamers could be decorated on the LNP surface through hybridization without conformational changes during the process (Fig. 2A) (23). Because of the complexity of the structure, mass spectrometry measurement was performed after each synthesis step to confirm the success of the synthesis and expected mass/charge ratio value was obtained for each step (fig. S6). Cryoelectron microscopy (cryo-EM) and high performance liquid chromatography (HPLC) analysis were performed to confirm successful encapsulation of IOX2 (Fig. 2, B and C). As expected, measurement of size and zeta potential showed that attachment of the aptamers increased the size and the negativity of the zeta potential following aptamer attachment (tables S1 and S2).

(A) Step-wise synthesis of aptamer-conjugated LNPs encapsulated with IOX2. (B) Aptamer-IOX2-LNPs under a cryo-EM. Yellow arrowheads indicate precipitation of IOX2, and red arrows indicate the conjugated aptamers. Scale bars, 100 nm. (C) HPLC chromatogram of IOX2-LNPs. (D) In vitro binding affinity of aptamer-IOX2-LNPs to mouse monocyte cell lines J774A.1 and RAW264.7, as well as mouse endothelial cell line SVEC. mAU, intensity of absorbance (in milli-absorbance units); RT, retention time; ns, not significant. (E) IVIS imaging of aptamer-IOX2-LNPs accumulation in the injured heart. The particles were labeled with the DiD lipophilic cyanine dyes. (F) Quantitative analysis of DiD-labeled aptamer-IOX2-LNPs in the injured heart using IVIS. ROI, region of interest. (G) Accumulation of aptamer-IOX2-LNPs in the infarct area under an intravital microscope. The aptamer-LNPs were labeled with the DiD lipophilic cyanine dyes. Scale bars, 100 m. (H) Biodistribution of aptamer-IOX2-LNPs in organs. One-way ANOVA with a Tukey adjustment was used to analyze data in (F). Two-way ANOVA with a Tukey adjustment was used to analyze the data in (D) and (H). ****P < 0.0001, **P < 0.01, and *P < 0.05.

Following the success of obtaining aptamer-LNPs, we examined the interaction between the LNPs and monocytes. Time-lapse live cell imaging taken over the course of 90 min of incubation between S2 and J10 aptamers with the monocyte cell line RAW264.7 showed that although some of nanoparticles were internalized, most of them remained on the surface, which is expected. More J10-LNPs were also observed on the surface of monocytes compared to S2, which further supports our finding that J10 is a better monocyte-targeting aptamer (fig. S7A and movies S5 and S6). We also investigated whether the attachment of aptamer-LNPs affected monocyte function. We profiled the cytokines [interleukin-1 (IL-1), IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor] of LNP-, J10-, and J10-LNPtreated RAW264.7 monocyte cell line using quantitative PCR. The results showed no changes in the levels of these cytokines, indicating that the nanoparticles did not affect the function of or cause adverse side effects to the monocytes (fig. S7B).

Having successfully encapsulated IOX2 in the J10-decorated nanoparticles, we then examined the ability of J10-IOX2-LNPs to bind to monocytes in vitro and to use monocytes to target IR hearts in vivo. Flow cytometry analysis using DiD [The far-red fluorescent dye DiD (1,1-Dioctadecyl-3,3,3,3-Tetramethylindodicarbocyanine Perchlorate)]labeled J10- and S2-LNPs revealed that the binding of J10-decorated LNPs to monocytes was more effective than S2-LNPs and nondecorated LNPs, with minimal binding to endothelial cells in vitro (Fig. 2D). For the in vivo study, in vivo imaging system (IVIS) analysis showed a significant increase in fluorescence for DiD-labeled J10-IOX2-LNPs compared to phosphate-buffered saline (PBS) (background) and DiD-labeled S2-IOX2-LNPs, indicative of successful targeting of J10-decorated nanoparticles to the injured hearts (Fig. 2, E and F). Intravital imaging further confirmed higher J10-IOX2-LNPs accumulation in the infarct area, suggesting that the nanoparticles successfully reached the intended site (Fig. 2G). Biodistribution study of IOX2-loaded S2- and J10-LNPs (Fig. 2H) showed a significant increase in IOX2 retention in the heart for J10-LNPs 4 hours after injection, indicating that our J10 aptamer drug delivery system successfully increased drug delivery to the heart. To confirm that J10-IOX2-LNPs delivered the IOX2 cargo by hitchhiking on the surface of monocytes, we depleted the circulating monocytes in IR mice using clodronate liposomes (24) and injected the nanoparticles. Complete blood count confirmed the success of monocyte depletion (fig. S8A), while quantification of IOX2 content in the heart showed significant decrease in clodronate-treated mice (fig. S8B). This result proved that our J10 drug delivery platform hitchhiked on the surface of monocytes to reach the injured heart.

The therapeutic effect of IOX2-loaded nanoparticles was then examined in a murine model of myocardial IR injury. The mice were injected with three doses of S2- and J10-IOX2-LNPs at 5 hours, 1 day, and 2 days after IR injury (Fig. 3A). These time points were optimal for therapy because injections at 5 hours or 5 days after IR injury resulted in a similar IOX2 accumulation level (fig. S9). Because IOX2 prevents the degradation of HIF-1, which is up-regulated early after IR injury, early injection time points were chosen for the efficacy trial. Furthermore, because the enhanced permeability and retention effect diminishes after 24 hours (3), the fact that accumulation of IOX2 remained similar at 5 hours and 5 days further suggests that nanoparticle delivery was achieved by hitchhiking on the monocyte surface. This is also supported by our monocyte recruitment and circulating monocyte profiles (Fig. 1 and fig. S1), where the monocyte levels remained high within these time points. We then aimed to understand the drug release profile, by performing biodistribution studies of IOX2 in J10-IOX2-LNPtreated IR mice (fig. S10). The nanoparticle injection was performed 5 hours after IR injury, and the organs were collected at different time points (5 hours, 1 day, and 4 days) after injection. We found that accumulation of IOX2 was at the highest at day 1 after injection and decreased at day 4. This suggests that the body started to eliminate the nanoparticles and the drugs after 24 hours after administration.

(A) Experimental design for in vivo functional evaluation of aptamer-IOX2-LNPs in the mouse cardiac IR injury model. (B) The protein levels of HIF-1 after aptamer-IOX2-LNP treatment. (C) Terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay for detection of apoptosis in the injured heart after aptamer-IOX2-LNP treatment. The apoptotic index was defined as of the percentage of TUNEL+ cells in a field examined. DAPI, 4,6-diamidino-2-phenylindole; CTnl, cardiac troponin I. (D) Staining for -smooth muscle actin (-SMA) and isolectin IB4 (IB4) to examine the effects of J10-IOX2-LNPs on angiogenesis in the injured heart. WGA, wheat germ agglutinin. (E and F) Quantification of -SMA+ (E) and IB4+ (F) vessels in the injured heart after aptamer-IOX2-LNP treatment. G) The effects of aptamer-IOX2-LNPs on cardiac fibrosis on day 21 after IR injury. (H) Quantification of cardiac fibrosis after aptamer-IOX2-LNP treatment. LV, left ventricle. (I to P) The effects of aptamer-IOX2-LNPs on the heart function 21 days after IR injury, including ejection fraction (EF) (I), fraction shortening (FS) (J), end-systolic volume (ESV) (K), end-diastolic volume (EDV) (L), dP/dt maximum (dP/dt max) (M), dP/dt minimum (dP/dt min) (N), ESPVR (end-systolic pressure-volume relationship) (O), and EDPVR (end-diastolic pressure-volume relationship) (P). (Q) The effects of aptamer-IOX2-LNPs on the survival rate of a mouse cardiac IR model. One-way ANOVA with a Tukey adjustment was used for data analysis. The Kaplan-Meier method and the log-rank (Mantel-Cox) tests were used for construction and analysis of the survival curves in (Q). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Following the injection of S2- and J10-IOX2-LNPs, the hearts were collected for analysis. Western blot analysis showed that J10-IOX2-LNP treatment retained the HIF-1 protein level in the heart, which indicates that IOX2 successfully reached the heart and prevented the degradation of HIF-1. This, in turn, is indicative of a cardioprotective effect (Fig. 3B). On the other hand, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay showed reduced number of apoptotic cells, demonstrating that our treatment prevented cardiomyocyte loss (Fig. 3C). In addition, J10-IOX2-LNP treatment also augmented angiogenesis, which was shown by the increased staining of -smooth muscle actin (-SMA) for vessels and isolectin B4 (IB4) for capillaries (Fig. 3, D to F). Trichrome staining of three levels of the heart on day 21 after IR injury showed that the J10-IOX2-LNP group had a significant reduction in infarct size compared to the controls, demonstrating better healing of the myocardium (Fig. 3, G and H). The results thus far indicated a better cardiac performance, which we then proved through echocardiography and cardiac catheterization experiments, which revealed that the J10-IOX2-LNP group showed significant improvement in all cardiac parameters in comparison to the control groups at day 21 (Fig. 3, I to P, and fig. S11).

To ensure the safety of our platform, we examined the hepatotoxicity [aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)] and nephrotoxicity [blood urea nitrogen (BUN) and CREA] of J10-IOX2-LNPs through serum analysis, all of which fell within the level of healthy animals (fig. S12, A to E). Histology analysis of the liver and kidneys was also performed, which showed no abnormalities (fig. S12F). All of these results combined showed that in the murine model of myocardial IR injury, our nanoparticles successfully targeted the injured hearts, resulting in improved cardiac functions, reduced infarct size, augmented angiogenesis, and, overall, prolonged survival of the mice (Fig. 3Q) without causing adverse side effects to the liver, kidneys, and monocytes.

Following the success of IR injury treatment with our J10 aptamer delivery platform, we then continued our investigation using this platform to treat PDAC mice. Gemcitabine, a drug used for pancreatic cancer treatment, was encapsulated into the nanoparticles using a passive loading method (table S3). The encapsulation success was confirmed by cryo-EM and by exploiting the presence of nuclear magnetic resonance (NMR)active 19F nuclei in gemcitabine using 19F NMR spectroscopy (2), as well as by HPLC (Fig. 4, A to C). A cytotoxicity assay confirmed that the gemcitabine toxicity to the tumor cells was retained following encapsulation (Fig. 4D).

(A) The aptamer-Gem-LNPs under a cryo-EM. Yellow arrowheads indicate precipitation of gemcitabine, and red arrows indicate the conjugated aptamers. Scale bars, 100 nm. (B) Representative 19F NMR spectrum of free and liposome-encapsulated gemcitabine. ppm, parts per million. (C) Representative HPLC chromatogram of free and liposome-encapsulated gemcitabine. (D) Cytotoxicity of free and liposome-encapsulated gemcitabine to cultured mouse pancreatic cancer (KPC) cell line. IC50, median inhibitory concentration. (E) In vitro targeting specificity of aptamer-Gem-LNPs against mouse monocyte and endothelial cell lines using flow cytometry. RAW264.7 and J774A.1 are the mouse monocyte cell lines; SVEC is a mouse endothelial cell line. (F to H) In vivo binding specificity of aptamer-Gem-LNPs to (F) monocytes, (G) lymphocytes, and (H) granulocytes. (I) Accumulation of aptamer-Gem-LNPs in mouse orthotopic pancreatic tumor determined with IVIS. The aptamer-Gem-LNPs were labeled with the DiD lipophilic cyanine dyes. (J) Quantification of gemcitabine accumulation in the mouse orthotopic pancreatic cancer using 19F NMR. Two-way ANOVA with a Tukey adjustment was used for data analysis in (E), and mixed-effects analysis was used to analyze data in (J). One-way ANOVA with a Tukey adjustment was used for data analysis in (F) to (I). *P < 0.05, **P < 0.01, and ***P < 0.001.

Having successfully encapsulated gemcitabine, we then examined the ability of J10-gemcitabine-LNPs (J10-Gem-LNPs) to selectively bind to monocytes in vitro and to deliver the cargo into the tumor site in vivo. Flow cytometry analysis using DiD-labeled J10- and S2-LNPs revealed that J10-Gem-LNPs were able to bind to monocytes more efficiently than to S2-LNPs and nondecorated LNPs, with minimal binding to endothelial cells in vitro (Fig. 4E). This was also confirmed in vivo through flow cytometry, whereby a preferential binding to monocytes but not to lymphocytes or granulocytes was observed (Fig. 4, F to H, and figs. S13 to S15). IVIS analysis of excised tumor showed that after 24 hours of LNP administration, the highest accumulation of nanoparticles was found in J10 group (Fig. 4I). We then aimed to understand the release profile of gemcitabine, by quantifying the amount of gemcitabine in PDAC mice at different time points (6, 24, and 48 hours) after injection (Fig. 4J). Comparison of gemcitabine content between S2 and J10 groups showed a significant accumulation at 24 hours and a modest accumulation at 48 hours for J10 group. This suggests that the body started to eliminate the nanoparticles and the drugs after 24 hours after administration, which is in agreement with the release profile of J10-IOX2-LNPs in IR hearts. All of these findings indicate that gemcitabine-loaded nanoparticles were able to target the tumor, with J10-decorated nanoparticles having the highest efficacy. To confirm that J10-Gem-LNPs delivered the gemcitabine cargo by hitchhiking on the surface of monocytes, we repeated the circulating monocyte depletion experiment in PDAC mice using clodronate liposomes and injected the nanoparticles. Complete blood count confirmed the success of monocyte depletion (fig. S8C), while quantification of gemcitabine content in the tumor showed a significant decrease in clodronate-treated mice (fig. S8D). This result proved that our J10 drug delivery platform hitchhiked on the surface of monocytes to reach the tumor site. Last, we investigated the effects of accumulated concentration of gemcitabine on monocytes, which showed that monocyte viability was not affected, suggesting no adverse side effects (fig. S16).

The therapeutic consequence of increased accumulation of gemcitabine-loaded nanoparticles was assessed in a murine PDAC model (Fig. 5A). TUNEL assay and proliferation assay using Ki67 showed that treatment with J10-Gem-LNPs significantly increased tumor cell apoptosis and decreased tumor cell proliferation, respectively, compared to S2-Gem-LNPs (Fig. 5, B and C), indicating that the treatment successfully hampered the growth of the tumor. This was then confirmed by IVIS and functional magnetic resonance imaging (fMRI) monitoring, which showed greater tumor growth suppression in the J10 group, in agreement with the tumor weight at the day of death (Fig. 5, D to F). Furthermore, treatment of gemcitabine-loaded nanoparticles did not affect the body weight (Fig. 5G), and serum chemistry assessment for hepatotoxicity (AST, ALT, and ALP) and nephrotoxicity (BUN and CREA) showed no adverse effects in both liver and kidney functions (fig. S17), which overall indicates the safety of J10-Gem-LNPs. All of these results combined showed that in the murine model of PDAC, our nanoparticles successfully targeted the tumor site, resulting in increased tumor cell apoptosis, reduced tumor cell proliferation and growth, and, overall, prolonged survival of the mice (Fig. 5H).

(A) Experimental design for the functional evaluation of aptamer-Gem-LNPs in a mouse orthotopic pancreatic cancer model. (B) J10-Gem-LNPs caused apoptosis of pancreatic tumor cells in vivo. The apoptotic index was determined with TUNEL assay. Scale bars, 20 m. (C) J10-Gem-LNPs reduced proliferation of pancreatic tumor cells in vivo. The proliferation index was determined by the ratio of Ki67+ cells. Scale bars, 20 m. (D) J10-Gem-LNPs reduced pancreatic tumor size on day 29 after treatment. The pancreatic tumor sizes were determined with IVIS to detect the luciferase activity of the mouse KPC cell line. (E) The J10-Gem-LNPs reduced pancreatic tumor size under MRI. (F) Quantification of orthotopic pancreatic tumor size harvested from mice treated with PBS, gemcitabine, Gem-LNPs, S2-Gem-LNPs, and J10-Gem-LNPs. (G) The effects of aptamer-Gem-LNPs on the body weight of the mouse orthotopic pancreatic cancer model. (H) J10-Gem-LNPs improved the survival rate of the mouse orthotopic pancreatic cancer model. (I) Effects of aptamer-Gem-LNPs on liver metastatic tumor volume under MRI. (J) Effects of aptamer-Gem-LNPs on the size of liver metastatic tumor on day 32 after treatment using IVIS. (K) Effects of aptamer-Gem-LNPs on the survival rate of mouse with liver metastatic tumors. Data in (B), (C), and (I) were analyzed with unpaired Students t test. One-way ANOVA with a Tukey adjustment was used for data analysis in (D) to (F) and (J). The data in (G) were analyzed with the two-way ANOVA with a Tukey adjustment. The survival curves in (H) and (K) were constructed with the Kaplan-Meier method and analyzed with the log-rank (Mantel-Cox) test. *P < 0.05, **P < 0.01, and ***P < 0.001.

As one of the most common metastatic site for pancreatic cancer is the liver, we further examined the therapeutic efficacy of our nanoparticles using a murine model of pancreatic cancer with liver metastasis (25). The progression of the metastatic tumor growth on the liver was similarly suppressed in the J10 group, as shown by fMRI and IVIS measurements (Fig. 5, I and J). Ultimately, we found that the J10-Gem-LNP platform was also capable of targeting liver metastasis, resulting in increased survival of the mice (Fig. 5K), which is in agreement to the results we obtained for the IR and PDAC models.

Previously, we have developed an injectable nanogel and reloadable targeted nanoparticles to improve the treatment of ischemic diseases such as myocardial infarction and hind limb ischemia (26, 27). However, both strategies are too invasive. Methods that rely solely on the ability of the drugs or drug-loaded nanoparticles to extravasate from the circulation into diseased sites are vastly limited by the availability and permeability of the blood vessels surrounding the sites. Although the method developed in our study also relies on the circulatory system to some extent, the drug-loaded nanoparticles were able to leave the blood stream and penetrate into the diseased site. With this strategy, we were able to successfully increase the therapeutic efficacy of drugs used in treating both IR injury and PDAC, a result that otherwise could not have been achieved.

Our aptamer-based LNP targeting system can be synthesized and is not patient specific. This eliminates the necessity to freshly prepare targeting scaffolds and, in a clinical setting, enables the treatment of patients who are in need of immediate administration of therapeutics. We have shown that our aptamer is capable of selectively binding to both murine and human monocyte cell lines (Fig. 1I and fig. S4), although the binding to human monocytes is not as strong as that to murine monocytes. This is expected, because we performed the SELEX procedure using murine monocyte cell lines, taking into account the difference between human and murine monocytes; this disparity is to be expected. Our findings have shown that circulating monocytes can be used as a shuttle bus for drug delivery using the appropriate aptamer-based targeting scaffold. Aptamers that can bind selectively to human monocytes with good affinity can be developed by following our approach using human monocytes to produce human monocyte-specific aptamers and be used for translational medicine purposes.

We have shown that our aptamer-based targeting vehicle was able to treat myocardial IR injury; however, we are limited by the monocyte recruitment time point and the number of circulating monocytes, which are at their optimum 4 days after injury (Fig. 1B and fig. S1). This time point is not early enough for the delivery of early cardioprotective therapeutics, which should ideally be administered a few hours after the IR episode. Nevertheless, delivery of therapeutics that prevents the heart from suffering further damage can be successfully achieved using our delivery method.

Using the same delivery vehicle and strategy, we assessed the therapeutic efficacy of our method in the treatment of PDAC. PDAC is known to exhibit hypovascularity, which makes treatments with reliance on the circulatory system challenging and ineffective (28). Fortunately, the development of PDAC involves the recruitment of monocytes in its pathogenesis (29), which is the basis of our therapeutic strategy. Therefore, although our aptamer-based delivery method also relies on the circulatory system to reach the tumor site, the ability of the drug-loaded nanoparticles to attach to monocytes, leave the blood vessel, and penetrate through the dense stromal extracellular matrix along with the monocytes increased the efficiency of drug delivery. This was validated by the increased amount of gemcitabine that successfully reached the tumor site, reduced tumor size and weight, and prolonged survival rate. Nevertheless, clinically, it is difficult to determine how inflammatory the tumor is at the time of treatment and if the treatment remains effective if given when the tumors are smaller (earlier) or larger (later). More studies involving the in vivo delivery kinetics will be required to further elucidate the therapeutic time window of this drug delivery system.

Last, our drug delivery system is potentially useful for the treatment of pancreatic cancer with liver metastasis. Before the formation of metastasis, monocytes are recruited to the liver (30, 31), to support the growth and proliferation of the invading tumor cells, in the end resulting in metastasis. Our delivery system was also assessed for treating liver metastasis, and we have shown that it was also able to reduce the metastatic tumor volume and prolong the survival of the mice suffering from pancreatic cancer with liver metastasis.

Our delivery system has a lot of advantages. It can potentially be used to deliver a wide variety of therapeutics such as small interfering RNA, modified RNA, antisense oligonucleotides, and protein drugs. It can also be used as a drug delivery platform for other diseases that involve monocyte recruitment in their pathophysiology. Furthermore, it is easy to manufacture and is not patient specific, which can potentially be useful for translational purposes. The only shortcoming of our study is that we only treated the mice for a short period of time, and although we managed to improve the overall condition and survival of the mice, we did not cure them. Prolonged treatment using our delivery platform may improve the overall outcome, and therefore, future longer-term studies are warranted.

Male 8- to 10-week-old wild-type C57BL/6 J mice, weighing approximately 25 g, were used for all experiments, unless otherwise stated. All mice were purchased from BioLASCO or National Laboratory Animal Center, Taiwan. Mice were housed in a 12-hour day/night cycle with unlimited access to food and water. Homozygous B6.129(Cg)-Ccr2tm2.1Ifc/J (CCR2RFP/RFP) and B6.129P2(Cg)-Cx3cr1tm1Litt/J (CX3CR1GFP/GFP) mice were purchased from the Jackson laboratory, USA. Heterozygous CCR2RFP/+ and CX3CR1GFP/+ mice were generated from Institute of Biomedical Sciences, Academia Sinica, Taiwan. For both intravital imaging and monocyte profiling, 6- to 8-week-old CCR2RFP/+ mice were used, while 10- to 12-week-old CX3CR1GFP/+ mice were used for intravital imaging. All mouse experiments have been approved by Academia Sinica Institutional Animal Care and Use Committee.

Mice (8 to 10 weeks old) were anesthetized with Zoletil 50 (80 mg/kg; Virbac) and Rompun (3.5 mg/kg; Bayer) and given O2 via a tracheal tube on a 37C heating pad. The heart was accessed via left thoracotomy between the third and fourth ribs. The left anterior descending coronary artery was temporarily ligated with sutures 7-0 polypropylene through polyethylene-10 tubing for 45 min. Subsequently, polyethylene-10 tubing was removed to induce myocardial IR injury. The success of the surgery was evaluated by echocardiography on the following day.

For orthotopic tumor implantation, 5 105 live KPC cells suspended in 20 l of sterile PBS were administered to 6- to 8-week-old C57BL/6 J mice by intrapancreatic injection around 2 to 3 mm from the pancreas tail. For the PDAC liver metastasis model, injection of KPC cells was performed on day 10 after orthotopic implantation by injection of 5 105 live KPC cells suspended in 10 l of sterile PBS into the portal vein using a Hamilton syringe.

Lipid film (total mass, 35 mg) was prepared in a round-bottom flask by dissolving 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and DSPE-PEG2000 in chloroform and DSPE-PEG2000 linker and DiD in methanol (molar ratio, 45:50:0.047:0.003:0.005). Solvent was removed under reduced pressure at room temperature, and the lipid film was lyophilized overnight.

IOX2-LNPs was prepared following a previously reported method (32). Briefly, the dry film was hydrated with 1 ml of internal buffer (200 mM calcium acetate) to form multilayer vehicles (MLVs). After the thin film was completely dissolved, the size and lamilarity of MLV were reduced by 10 freeze-thaw cycles under vacuum using liquid nitrogen and a 65C water bath. It was then sonicated using a probe sonicator in total for 2 min through a series of 2-s sonication and 10-s pause. Following this, liposome solution was extruded through a 0.1-m polycarbonate membrane 20 times at 65C to obtain around 100-nm small unilamilar vehicle linkerLNP. Calcium acetate was removed using Sepharose CL-4B size exclusion column to establish the liposome cross membrane gradient. Then, IOX2 was incubated with liposome in a drug to a lipid molar ratio of 0.4 at 65C for 30 min. Unencapsulated IOX2 was removed by Sepharose CL-4B size exclusion column with PBS as the mobile phase. Linker-IOX2-LNPs were then hybridized with J10 and S2 aptamers separately through overnight incubation at 4C (linker:aptamer, 1:2.5). Free aptamer was removed by Sepharose CL-4B size exclusion column with PBS as the mobile phase.

For fabrication of Gem-LNPs, the dry film was hydrated by 1 ml of gemcitabine in PBS solution (75 mg/ml) to form MLV linkerGem-LNP. After the dry film was completely dissolved, the size of MLV was reduced by 10 freeze-thaw cycles under vacuum using liquid nitrogen and a 65C water bath. Linker-Gem-LNP was sonicated using a probe sonicator in total for 2 min through a series of 2-s sonication and 10-s pause. Linker-Gem-LNP was then extruded through a 0.1-m polycarbonate membrane 20 times at 65C and stored overnight at 4C. Linker-Gem-LNPs were purified using a Sepharose CL-4B size exclusion column with PBS as the mobile phase. Pure linker-Gem-LNPs were then hybridized with J10 and S2 aptamers separately through overnight incubation at 4C (aptamers:linker, 2.5:1), followed by purification using a Sepharose CL-4B size exclusion column with PBS as the mobile phase.

Following the encapsulation, the drug concentration was measured to be 0.0625 mg per mg/ml of lipid and 0.186 mg per mg/ml of lipid for IOX2 and gemcitabine, respectively. The dosages used for the in vivo experiments are 0.7 mg of IOX2/kg for three injections and 1.66 mg of gemcitabine/kg for three injections.

The multiphoton intravital imaging was performed following a published procedure (33). All animals were anesthetized by 1.5% isoflurane (Minrad) during the experiment. Injection of 100 l of 5 mM S2-IOX2-LNP and J10-IOX2-LNP was administered to IR day 1 CCR2RFP/+ mice for an hour, and then the infarct area was visualized by a multiphoton microscope (FVMPE-RS, Olympus). Because the fluorescence of DiD-labeled IOX2-LNP was quenched within seconds under multiphoton imaging, QD655s (20 l; Invitrogen) modified with S2 or J10 were injected to CCR2RFP/+ and CX3CR1GFP/+ mice to visualize J10-QD655stagged monocytes passing through the blood vessel.

GraphPad Prism 8 was used for all statistical analysis and graph generation. Statistical tests are described in the figure legends. For group analysis, one-way or two-way analysis of variance (ANOVA) with Tukeys multiple comparison tests was used. For survival analysis, deaths were recorded and used to generate Kaplan-Meier survival curves, which were compared using Mantel-Cox log-rank tests. IVIS images of tumor luminescence and nanoparticle fluorescence were quantified using Living Image 3.1 software. For tumor size quantification, MRI images were processed in Avizo using the measure tool. 19F NMR spectra acquisition was performed on Bruker TopSpin 2.1 and processed on Bruker TopSpin 2.1 or 4.0.2. Adjustments to immunofluorescence image brightness and contrast were made to improve visual clarity and were applied equally to all images within a series. Figures were assembled in Adobe Illustrator.

Acknowledgments: We would like to thank the aptamer core facility in the Institute of Biomedical Sciences (IBMS), Academia Sinica for Cell-SELEX assistance. We would also like to thank the IBMS Flow Cytometry Core facility for flow cytometry analysis and Y.-H. Chen and IBMS Animal Core staff for animal experiments. We thank Academia Sinica High-Field NMR Center (HFNMRC) for technical support. We also thank J.-H. Lin, P.-J. Lin, and S.-C. Ruan DVM for assistance with the animal experiments. Funding: This work was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2319-B-001-004, 108-2321-B-001-017, and 108-3111-Y-001-053), the National Health Research Institutes grant EX109-10907SI and the Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis (AS-KPQ-106-TSPA), the Thematic Research Program (AS-107-TP-L12), and the Summit Research Program (MOST 107-0210-01-19-01). HFNMRC is funded by the Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-108-112). Author contributions: S.-S.H. and K.-J.L. designed and performed experiments and contributed to data analysis, manuscript, and figure preparation. H.-C.C. contributed to the data analysis, discussion, and figure design. R.P.P. performed experiments and contributed to the discussion and manuscript preparation. C.-H.H., O.K.C., S.-C.H., and C.Y.B. performed experiments. C.-B.J. and X.-E.Y. contributed to the IOX2-liposome fabrication. D.-Y.C. and C.W.K. performed the intravital imaging. T.-C.C. established the orthotopic pancreatic cancer model. L.-L.C. drew the schematic illustration. J.J.L. and T.J.K. contributed to the discussion. P.C. managed the intravital imaging. Y.-W.T. contributed to the discussion of PDAC experiments. H.-M.L. managed the liposome fabrication and characterization. P.C.-H.H supervised and managed the project. Competing interests: T.J.K. serves as a consultant for Fujifilm Cellular Dynamics Incorporated. P.C.-H.H., S.-S.H., K.-J.L., and H.-C.C. have patent provisional applications (US 2020/63030674 and US 2020/63030555) related to the use of aptamer-based drug delivery for treatment of heart diseases and cancer. The patent provisional applications were filed by Academia Sinica. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. For patent and tech transfer concerns, the raw and analyzed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Additional data related to this paper may be requested from the authors.

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