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

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

The authors report no conflicts of interest.

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

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

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

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

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).


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 or visit to learn more, and follow Crusoe on Linkedin and Twitter.

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

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Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer – Science Advances


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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Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer - Science Advances

Recommendation and review posted by Alexandra Lee Anderson

Scientists Pinpoint the Spots of Early Prion Protein Deposition in the Retina – Gilmore Health News

What is prion disease?

Prion diseases are a type of neurodegenerative disorder that is produced by the accumulation of abnormal proteins in the brain. Prion disease predominantly affects the brain, but it can also attack the eyes, especially the light-sensitive photoreceptors called cones and rods which are present in the retina, and other organs. These are steadily deteriorating and typically deadly diseases of the brain and can occur in people as well as some other mammals. Examples: mad cow disease in cattle, Creutzfeldt-Jakob disease in people, chronic wasting disease in deer, elk, and moose, and bovine spongiform encephalopathy in cattle.

Prion Infected Retina. Image Courtesy of NIH

Read Also: Creutzfeldt-Jakob Disease: A Lab Technician Gets Disease 7 Years After Accidental Cut

A recent study done by scientists at the National Institutes of Health states that the initial eye injury from prion disease occurs in the cone photoreceptor cells, especially in the cilia and the ribbon junctions. The researchers say, their discovery may provide understanding on human retinitis pigmentosa, an inherited disorder with closely related photoreceptor degradation advancing into blindness. The understanding of how prion diseases develop in the eyes can aid scientists to look for strategies to steady the growth of prion diseases.

In their study, the researchers, from NIHs National Institute of Allergy and Infectious Diseases at Rocky Mountain Laboratories in Hamilton, Montana, used research mice diseased with scrapie, a prion disease routine to sheep and goats. Scrapie is nearly associated with human prion diseases, Creutzfeldt-Jakob disease (CJD).

Read Also: Alzheimers: What If It Is Similar to Mad Cow Disease?

The scientists discovered the accumulation of a lump of prion protein was seen first in cone photoreceptors next to the cilia, pipe-like formation needed for transferring molecules between cellular sections with help of the confocal microscope. The study suggests that by obstructing the movement through cilia, these clumps may layout a key early process by which prion infection particularly smashes photoreceptors. Relatable findings were seen in rods as well.Exactly before the destruction of ribbon synapses (specialized neutron links present in the eye and ear neural pathways) and end of photoreceptors, there was an accumulation of prion protein in these structures.

The findings from this study were unique and were never observed before. The association between prion protein and retinal injury is probably present in all prion-vulnerable species, as well as humans.

There are other kinds of declining disorders that are also distinguished by abnormal folding of self-proteins, such as Alzheimers and Parkinsons diseases. The scientists are looking to investigate if related findings take place in the retinas of these people.

Read Also: An Artificial Retina to Restore Sight Could Soon Become a Reality

Prion-induced photoreceptor degeneration begins with misfolded prion protein accumulation in cones at two distinct sites: cilia and ribbon synapses

Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients

Scientists Pinpoint the Spots of Early Prion Protein Deposition in the Retina - Gilmore Health News

Recommendation and review posted by Alexandra Lee Anderson

Targeting oncoproteins with a positive selection assay for protein degraders – Science Advances


To develop a positive selection assay for protein degraders, we made a bicistronic lentivirus encoding (i) a POI fused to a modified version of deoxycytidine kinase (hereafter called DCK*) that converts the non-natural nucleoside 2-bromovinyldeoxyuridine (BVdU) into a poison (4) and (ii) green fluorescent protein (GFP). We reasoned that GFP could be used to mark reporter-positive cells, to FACS (fluorescence-activated cell sorting) sort for cells with the desired reporter mRNA levels, and to count cells in multiwell plate assays. In our initial proof-of-concept experiments, we used this virus to create 293FT cells expressing the IMiD target IKZF1 (1, 2, 5) fused to DCK* and compared them to cells expressing unfused DCK* or unfused IKZF1 (Fig. 1, A and B). As expected, the IMiD pomalidomide (POM) down-regulated DCK*-IKFZ1 and IKZF1 but not DCK* (Fig. 1B). We also confirmed that 293FT cells expressing DCK*-IKZF1 or unfused DCK* were more sensitive to BVdU than 293FT cells expressing IKZF1 alone or infected with an empty vector (EV) (Fig. 1C). The increased BVdU sensitivity of the DCK* cells relative to the DCK*-IKZF1 cells is likely explained by the higher protein levels of DCK* compared to DCK*-IKZF1 (Fig. 1B). Similar results were observed with cells expressing DCK*-K-Ras (G12V), DCK*-Cyclin D1, DCK*-FOXP3, and DCK*-MYC, indicating that DCK* remains active when fused to a variety of proteins (fig. S1). POM increased the BVdU median effective concentration (EC50) of cells expressing DCK*-IKZF1 but not of cells expressing DCK* (Fig. 1D).

(A) Vector schematic. DCK*, variant deoxycytidine kinase with Ser74Glu, Arg104Met, and Asp133Ala substitutions; V5, V5 epitope tag; GGS, Gly-Gly-Ser spacer; IRES, internal ribosomal entry site. (B) Immunoblot analysis of 293FT cells infected with the lentiviral vectors depicted in (A) and then treated with 1 or 10 M POM, as indicated by the triangles, for 24 hours. (C and D) Relative survival of 293FT cells infected with the lentiviral vectors depicted in (A) and then treated with the indicated concentrations of BVdU for 4 days. In (D), cells were also treated with 1 M (POM) starting 24 hours before BVdU was added. n = 3 biological replicates. (E and F) Number of GFP-positive 293FT cells infected to produce DCK* (E) or DCK*-IKZF1 (F) using the vectors in depicted in (A) and then treated with indicated concentrations of POM and BVdU in 384-well plate format. POM was added 24 hours before treatment with BVdU for 4 days. n = 4 biological replicates. (G) Immunoblot analyses of cells treated as in (E) and (F). (H) Fluorescence data of 384-well plate containing 293FT cells expressing DCK*-IKZF1 treated with DMSO (columns 1 to 11 and 24) or 1 M POM (columns 12 to 23), followed 24 hours later by the addition of 100 M BVdU for 4 days (columns 1 to 24).

Next, we seeded either the DCK*-IKZF1 cells or DCK* cells in 384-well plates and treated the wells with increasing amounts of POM or with dimethyl sulfoxide (DMSO). We added BVdU 24 hours later and measured cell viability 4 days thereafter by measuring the number of GFP-positive objects per well. POM again promoted the survival of the DCK*-IKZF1 cells, but not the DCK* cells, over a range of POM and BVdU concentrations (Fig. 1, E to G). In anticipation of using this assay for a high-throughput screen, we next seeded the DCK*-IKZF1 cells in 384-well plates and treated half the wells with POM and half the wells with DMSO, followed 24 hours later by BVdU (Fig. 1H). Measuring GFP-positive objects 4 days later produced a favorable Z value (0.7) for this assay.

Encouraged by these findings, we did a pilot screen with 293FT cells expressing DCK*-IKZF1 or unfused DCK* grown in 384-well plates and a library of ~2000 bioactive compounds, which included lenalidomide (LEN) and POM (Fig. 2, A to C). Each well received a different compound at a concentration of approximately 10 M by pin transfer, followed the next day by BVdU. BVdU was added at 100 M to the DCK*-IKZF1 cells and at 10 M to the DCK* cells to achieve comparable cell killing despite the higher levels of DCK* relative to DCK*-IKZF1 (fig. S2). Four days thereafter, the GFP fluorescence for each well was measured and converted to a z score based on the GFP fluorescence values for the other wells on its plate. LEN and POM scored positively (z > 2) in the DCK*-IKZF1 screen but not the DCK* screen (Fig. 2, B to E). Some compounds promoted the survival of both DCK* cells and the DCK*-IKZF1 cells, including compounds that interfere with BVdU uptake (e.g., dipyridamole) (6, 7) or incorporation into DNA (e.g., thymidine) (compare Fig. 2, B and C). Such assay positives could be largely eliminated by subtracting the DCK* z score for each chemical from its DCK*-IKZF1 z score (Fig. 2F). For comparative purposes, we also did a screen with the same 2000 bioactive compound collection using 293FT cells expressing a bicistronic mRNA encoding (i) an IKZF1Firefly luciferase (Fluc) fusion and (ii) Renilla luciferase (Rluc), using a decrease in the Fluc/Rluc ratio to identify IKZF1 degraders (Fig. 2, G to I, and fig. S3) (2). As expected for such a down assay, this screen underperformed the DCK*-IKZF1 up screen with respect to both signal to noise and the number of false positives, which included compounds that inhibit Cap-dependent translation (e.g., VX-11e or BIX02565) (810). Compounds that nonselectively inhibit transcription, translation, or protein folding would predictably be especially problematic for Fluc fusions with shorter half-lives than the Rluc internal control. Notably, the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide did not promote the survival of the DCK*-IKZF1 cells at any concentration tested (fig. S4).

(A) Scheme for positive selection protein degradation assay. (B and C) Representative fluorescence data of 384-well plates containing 293FT cells expressing DCK* (B) or DCK*-IKZF1 (C) treated with compounds in the Selleck BioActive Library (one compound per well), followed 24 hours later by the addition of BVdU at the EC85 (10 and 100 M, respectively) for 4 days. BVdU was omitted in column 1. Columns 23 and 24 contained 10 M POM and 12.5 M dipyridamole (DiP), respectively. Library wells containing POM and DiP are indicated by the red and white arrows, respectively. (D and E) Z-distribution of GFP fluorescence of DCK* cells (D) and DCK*-IKZF1 cells (E) screened with the full Selleck BioActive Library. LEN and POM are indicted by the blue circle and red triangle, respectively. n = 2 biological replicates. (F) Corrected z scores obtained by subtracting z scores in (D) from z scores in (E). (G) Scheme for negative selection screening using the dual-luciferase reporter assay. (H and I) Z scores of Fluc/Rluc ratio of 293FT IKZF1-Fluc-IRES-Rluc cells after screening with the Selleck BioActive Library for 8 hours (H) or 4 days (I). n = 2 biological replicates.

As one way to minimize false positives, we seeded 384-well plates with a 1:1 mixture of 293FT cells expressing either (i) DCK*-IKZF1 and GFP or (ii) DCK* and TdTomato (Fig. 3A). Both POM and dipyridamole increased the number of GFP-positive cells, but dipyridamole was readily identified as a false positive by examining the TdTomato fluorescence channel (Fig. 3B). We then repeated these experiments in 384-well plate format, exposing the cells to 10 different concentrations of a small library of approximately 100 analogs of POM that we had synthesized, which included the known IKZF1 degraders LEN, POM, and avadomide (MI-2-65) (11) and several uncharacterized IMiD-like molecules from the literature (12) (Fig. 3C and tables S1 and S2). This library was generated to test whether our assay could correctly identify the known IKZF1 degraders and identify additional IKZF1 degraders made by alternative diversification of the aryl moiety of POM. LEN, POM, and avadomide all scored in our assay (Fig. 3C). In addition, several previously uncharacterized compounds, including MI-2-61 and MI-2-197, appeared to be at least as potent as POM in this screen and in confirmatory immunoblot assays (Fig. 3, C to F, and fig. S5). Our screen also correctly classified compounds that did not down-regulate IKZF1 in immunoblot assays, including some (e.g., MI-2-192 and MI-2-118) that still bound to cereblon in biochemical assays (fig. S5).

(A) Scheme for in-well GFP/TdTomato competition assay. 293FT cells were infected to produce DCK*-IKZF1 and GFP or DCK* and TdTomato using bicistronic vectors analogous to those depicted in Fig. 1A. (B) Top: Heatmap of the fold change (relative to treatment with DMSO) of GFP fluorescence of a 1:1 mixture of GFP-positive DCK*-IKZF1 and TdTomato-positive DCK* cells treated with 3.125, 6.25, 12.5, or 25 M POM or dipyridamole or with vehicle (DMSO) and followed 1 day later by the addition of 100 M BVdU for 4 days. Bottom: Heatmap of the fold change (relative to treatment with DMSO) of the ratio of GFP fluorescence to TdTomato fluorescence of the cells treated in (A). n = 2 biological replicates. (C) Heatmap of the fold change (relative to treatment with DMSO) of the ratio of GFP to TdTomato fluorescence of a 1:1 mixture of GFP-positive DCK*-IKZF1 and TdTomato-positive DCK* cells treated with 1.3 nM, 3.8 nM, 11.4 nM, 34 nM, 102 nM, 310 nM, 920 nM, 2.78 M, 8.33 M, and 25 M of the indicated IMiDs, as indicated by the triangles, or with vehicle (DMSO), and followed 1 day later by the addition of 100 M BVdU for 4 days. n = 2 biological replicates. (D) Immunoblot analysis of 293FT cells lentivirally transduced to express IKZF1-V5 and treated with the indicated IMiD derivatives for 24 hours using the same concentration range as in (C). (E) Structures of POM and IMiD MI-2-61. (F) Quantification of immunoblot data in (D); n = 2 biological replicates.

To begin looking for non-IMiD IKZF1 degraders, we screened ~546 metabolic inhibitors and anticancer drugs at 10 different concentrations using the DCK*-IKZF1 293FT cells in 384-well plate format (tables S3 and S4) (13). In parallel, we counterscreened against unfused DCK* cells. Spautin-1 (14), like POM, promoted the survival of the DCK*-IKZF1 cells, but not the DCK* cells, in a dose-dependent manner (Fig. 4, A to D). We confirmed that Spautin-1 down-regulated DCK*-IKZF1 and V5-tagged exogenous IKZF1 but not DCK* (Fig. 4E). IKZF1-V5 was among the 100 most down-regulated proteins after 24 hours of Spautin-1 treatment, as determined by quantitative mass spectrometry proteomics (fig. S6 and table S5). Until the direct target of Spautin-1 linked to IKZF1 turnover is known, it is impossible to know how many of these changes in protein abundance are direct versus indirect and on-target versus off-target. Notably, Spautin-1, unlike POM, down-regulated IKZF1 in cells lacking cereblon (Fig. 4F).

(A) Chemical structure of Spautin-1. (B) GFP fluorescence of DCK*-IKZF1 and DCK* 293FT cells treated with ranolazine, Spautin-1, and resveratrol at concentrations of 25 M, 8.33 M, 2.78 M, 920 nM, 310 nM, 102 nM, 34 nM, 11.4 nM, 3.8 nM, and 1.3 nM, as indicated by the triangle, followed 24 hours later by the addition of BVdU at the EC85. Shown for comparison are cells treated with POM (10 M) or dipyridamole (DiP) (12.5 M) before adding BVdU. n = 2 biological replicates. (C and D) Quantification of GFP fluorescence from (B) for Spautin-1 (C) and for an analogous titration with POM (D). (E) Immunoblot analysis of 293FT cells infected with lentiviruses as in Fig. 1A and treated with the indicated concentrations of Spautin-1 for 24 hours. (F) Immunoblot analysis of isogenic 293FT CRBN +/+ and CRBN / cells transduced to express IKZF1-V5 and treated with the indicated concentrations of Spautin-1 or POM (1 M) for 24 hours. (G) Immunoblot analysis of 293FT cells stably expressing IKZF1-V5 and simultaneously treated with MLN7243 (1 M), MLN4924 (1 M), MG132 (1 M), Spautin-1 (10 M), or POM (1 M) for 24 hours as indicated. (H and I) Immunoblot (H) and RT-qPCR (I) analysis of KMS11 multiple myeloma cells treated with indicated concentrations of Spautin-1 or POM (1 M) for 24 hours. n = 3 biological replicates.

Spautin-1 reportedly suppresses autophagy by inhibiting the USP10 and USP13 deubiquitinases (14). IKZF1 protein levels were not decreased after small interfering RNAmediated down-regulation of USP10, alone or in combination with USP13 (fig. S7A), and Spautin-1s ability to down-regulate IKZF1 was not altered when one or both of these proteins were suppressed (fig. S7, B and C). Moreover, Spautin-1 down-regulated IKZF1 in 293FT cells in which autophagy was disabled by CRISPR-Cas9mediated disruption of ATG7, Beclin1, or FIP200 (fig. S8).

In contrast, down-regulation of IKZF1 by Spautin-1 was blocked by compounds that inhibit either the E1 ubiquitin activating enzyme or the proteasome (Fig. 4G). Down-regulation of IKZF1 by Spautin-1 was not, however, blocked by an inhibitor of neddylation, which is required for cullin-dependent ubiquitin ligases [e.g., the cereblon-containing ubiquitin E3 ligase that is coopted by the IMiDs (1, 2, 5)] (Fig. 4G). Down-regulation of exogenous IKZF1 by Spautin-1 requires the IKZF1 N-terminal region containing IKZF1s first zinc finger domain (ZF1) but not the IKZF1 zinc finger domain (ZF2) targeted by the IMiDs (fig. S9, A and B) (15, 16). The down-regulation of the N terminus of IKZF1 was similarly blocked by compounds that inhibit either the E1 ubiquitin activating enzyme or the proteasome but not by inhibitors of neddylation (fig. S9C). Preliminary structure-activity relationship studies identified both active and inactive Spautin-1 derivatives (fig. S10), suggesting that down-regulation of IKZF1 by Spautin-1 reflects a specific protein-binding event and that Spautin-1s potency and specificity can be optimized further.

The experiments described above implied that Spautin-1 posttranscriptionally regulates IKZF1. Nonetheless, Spautin-1 also suppressed exogenous IKZF1 mRNA levels in 293FT cells (fig. S11). However, Spautin-1 suppressed endogenous IKZF1 protein levels in KMS11 and L363 myeloma cells at concentrations that minimally suppressed IKZF1 mRNA levels (Fig. 4, H and I, and fig. S12, A to C). Spautin-1 did not down-regulate IKZF1 in all myeloma cells tested (fig. S12, D and E). The biochemical basis for this variability is not clear.

Notably, down-regulation of IKZF1 by Spautin-1 occurs much more slowly than with IMiDs, suggesting that its effect on IKZF1 is indirect (fig. S13). Nonetheless, its ability to score in a positive selection assay, as well as its inability to down-regulate IKZF1 in some myeloma lines, suggests that it is not broadly toxic at concentrations that down-regulate IKZF1. We are currently seeking the direct Spautin-1 target linked to IKZF1 turnover using genetic and biochemical tools.

One advantage of positive selection assays is their enablement of pooled screens. Our positive selection assay, however, uses a suicide gene. Some suicide genes cause bystander killing that could confound their use in pooled screens. In pilot studies, however, we confirmed that DCK*-IKZF1 cells rapidly outgrew DCK* cells in cocultures treated with IMiDs and BVdU (fig. S14A) and that the DCK* single guide RNA (sgRNA) was rapidly and specifically enriched relative to the control sgRNA in Cas9-positive 293FT cells expressing either DCK*-IKZF1 or DCK*-FOXP3 and then treated with BVdU (fig. S14, B and C). Therefore, bystander killing is negligible in this system.

To begin to address the general utility of our methodology, as well as its ability to function in a pooled format, we next did experiments with ASCL1 in place of IKZF1. ASCL1 is an undruggable lineage-specific transcription factor that is required for survival in many small cell lung cancers (SCLCs) and neuroblastomas (1719). We made Jurkat T cells that express Cas9 and either (i) DCK*, (ii) the neural/neuroendocrine lineagespecific transcription factor ASCL1, (iii) DCK*-ASCL1, or (iv) ASCL1-DCK* (Fig. 5A and fig. S15A). Jurkat cells were chosen because they are easily grown and expanded in suspension cultures. ASCL1-DCK* was chosen for further study because we could not generate cells producing high levels of DCK*-ASCL1 (fig. S15A). We first confirmed that ASCL1-DCK* expression sensitized Jurkat cells to BVdU and that this was partially reversed after down-regulating the fusion with ASCL1 sgRNAs (Fig. 5, A and B, and fig. S16, A and B). Cas9 expression was also slightly attenuated in the ASCL1-DCK* cells over time for unclear reasons (Fig. 5A). Nonetheless, these cells efficiently edited a GFP-based reporter of Cas9 activity within 10 days of receiving a GFP sgRNA (fig. S15, B and C). Next, we infected the ASCL1-DCK* and DCK* cells with a lentiviral sgRNA library targeting 788 genes (seven sgRNAs per gene) that encode druggable proteins (table S6). Ten days later (to allow time for gene editing), the cells were split and grown in the presence of 200 or 500 M BVdU for an additional 2 weeks (fig. S16C). We then determined sgRNA abundance by next-generation sequencing of genomic DNA and analyzed relative enrichment of sgRNAs compared to the time point before BVdU treatment (fig. S16D). We identified multiple sgRNAs against CDK2 that were markedly enriched at both BVdU concentrations in the ASCL1-DCK* cells but not the DCK* cells (Fig. 5C, fig. S16, D and E, and table S7).

(A) Immunoblot analysis of Jurkat cells first infected to express Cas9 and then superinfected to express exogenous ASCL1, DCK*, or the ASCL1-DCK* fusion. NCI-H69 cells are included as a benchmark for ASCL1 endogenous expression. (B) Growth inhibition (%), based on viable cell numbers relative to untreated controls, of the indicated cell lines from (A) treated with BVdU for 6 days. n = 2 biological replicates. (C) Hypergeometric analysis of BVdU positive selection CRISPR-Cas9 screen on day 25 relative to day 10 (early time point before BVdU treatment) of ASCL1-DCK* Cas9 Jurkat cells treated with 500 M BVdU. n = 2 biological replicates. (D) Quantification of fold change in mCherry:BFP ratio after 18 days of 500 M BVdU or DMSO (0) treatment of ASCL1-DCK* Cas9 Jurkat cells expressing the indicated sgRNAs and mCherry or a nontargeting control sgRNA and blue fluorescent protein (BFP) (initially mixed 1:3). n = 3 biological replicates. (E) Immunoblot and (F) RT-qPCR analysis of ASCL1-DCK* Cas9 Jurkat cells superinfected to express the indicated sgRNAs. n = 4 biological replicates. (G) Immunoblot analysis of Jurkat cells first infected with a lentivirus to stably express exogenous ASCL1, then infected with Dox-inducible (DOX-On) sgRNA-resistant CDK2 wild-type (WT) or CDK2 kinase-dead (KD) mutant, and lastly superinfected with a CDK2 or nontargeting sgRNA. Following superinfection with the sgRNA lentiviruses, cells were grown in DOX to maintain exogenous CDK2 expression. n = 4 biological replicates. Exo, exogenous CDK2; Endo, endogenous CDK2. Error bars represent SD. ns, nonsignificant; *P < 0.05; ***P < 0.001; ****P < 0.0001.

In validation studies, ASCL1-DCK* Jurkat cells expressing CDK2 sgRNAs outcompeted ASCL1-DCK* cells expressing control sgRNAs in the presence of BVdU but not in the presence of DMSO (Fig. 5D and fig. S16F). CDK2 sgRNAs also posttranscriptionally down-regulated ASCL1-DCK* protein levels in the Jurkat cells (Fig. 5, E and F) and endogenous, unfused, ASCL1 in human SCLC lines (NCI-H1876 and NCI-H2081) (Fig. 6, A and B, and fig. S18, A and B). Down-regulation of exogenous ASCL1 in Jurkat cells treated with a CDK2 sgRNA was rescued by an sgRNA-resistant CDK2 complementary DNA (cDNA) encoding wild-type CDK2 but not kinase-dead CDK2 (Fig. 5G). The kinase-dead CDK2 was, however, produced at slightly lower levels, presumably because it is less stable or because of its known dominant-negative effects due to cyclin sequestration (2022).

(A) Immunoblot and (B) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. (C and E) Immunoblot and (D and F) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. (G) Immunoblot analysis and (H) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 g/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001.

CDK2 has been well recognized as a potential anticancer target. The development of selective small-molecule CDK2 inhibitors, however, has been hampered by their off-target effects on other CDK family members, especially the broadly essential kinase CDK1. We verified that well-established CDK2 inhibitor dinaciclib (23) down-regulated both ASCL1 protein and mRNA levels (fig. S17, A to D), potentially due to its polypharmacological activity on both CDK2 and other CDKs such as CDK9 (23, 24). We obtained, however, two small-molecule CDK2 degraders (TMX-2138 and TMX-2172) that more selectively target CDK2 through recruitment of cereblon (25). Both of these compounds down-regulated ASCL1 protein levels in both human (NCI-H1876 and NCI-H1092) and mouse (97-2 and 188) SCLC lines (Fig. 6, C to F, and fig. S18, C to F). For unclear reasons, ASCL1 was down-regulated more rapidly in the mouse lines than in the human lines. We focused on TMX-2172 because TMX-2138 also suppressed ASCL1 mRNA levels in the mouse cells (Fig. 6F and fig. S18F). TMX-2172 decreased the half-life of ASCL1 protein (Fig. 6, G and H), consistent with posttranscriptional regulation of ASCL1 by CDK2.

We conducted our screens in IKZF1-independent 293FT cells rather than IKZF1-dependent myeloma cells and in ASCL1-independent Jurkat cells rather than in ASCL1-dependent SCLC cells in an attempt to preserve positive selection. It is possible, however, that some degradation mechanisms will be highly context dependent and restricted to the therapeutic target cell of interest. We also anticipate that some DCK* fusion proteins will not be functional due to steric or conformational effects. This might be remedied by fusing DCK* to the alternative POI terminus (N-terminus versus C-terminus), by exploring different linkers, or using alternative suicide proteins.

IMiDs are important multiple myeloma drugs, but loss of cereblon has emerged as an important mechanism of IMiD resistance (2628). Identification of Spautin-1s mechanism of action could eventually lead to drugs for circumventing this problem.

ASCL1 is a sequence-specific DNA binding transcription factor that would classically be deemed undruggable and serves as a lineage addiction oncoprotein in neural crestderived tumors, such as SCLCs and neuroblastomas (1719, 29). Genetic studies in Xenopus indicate that CDK2 regulates ASCL1 function and that ASCL1 contains multiple potential CDK2 phosphorylation sites that prevent it from inducing neuronal differentiation (30, 31). CDK2 is a potential dependency in some neuroblastomas (3234). CDK2 and N-MYC drive the accumulation of phosphorylated ASCL1 in undifferentiated neuroblastomas (31). Conversely, loss of CDK2 activity, such as through retinoic acidmediated induction of p27 or small-molecule inhibitors, is associated with neuroblastoma differentiation and decreased tumor formation (3237). It will be important to determine how, mechanistically, CDK2 regulates ASCL1 turnover. In particular, we have not yet shown that the regulation of ASCL1 by CDK2 is direct. Nonetheless, our study provides further support for CDK2 as a potential therapeutic target in SCLC and neuroblastoma.

The discovery that the IMiDs reprogram the cereblon ubiquitin E3 ligase for therapeutic benefit has galvanized interest in identifying compounds that can degrade, directly and indirectly, otherwise undruggable proteins. Sometimes, one can engineer heterobifunctional degrader molecules consisting of a POI-binding moiety, a linker, and a ubiquitin-ligase recruitment moiety (38). This approach requires a ligand with suitable binding affinity for the POI, and identifying a successful linker often requires multiple iterations of trial and error. Moreover, this approach fails to harness the many other ways a chemical could directly or indirectly degrade a protein, such as by inhibiting a deubiquitinating enzyme, displacing an interacting protein, or altering protein folding or subcellular localization. A trivial way to down-regulate proteins, especially those with naturally rapid turnovers, is to poison transcription or translation. The screening methodology described here should facilitate the characterization of designer degraders as well as enable mechanism-agnostic searches for compounds and targets that regulate the abundance of previously undruggable proteins.

293FT cells were originally obtained from the American Type Culture Collection (ATCC). 293AD cells were from Cell Biolabs. 293FT CRBN / cells were made by CRISPR-Cas9 editing (see below). 293FT and 293AD cells were maintained in Dulbeccos minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml). KMS11, KMS34, MM.1S, and L363 human multiple myeloma cells [gift of K. Anderson (Dana-Farber Cancer Institute)] and Jurkat cells (obtained from ATCC in September 2016) were maintained in RPMI medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml). NCI-H1876 (obtained in November 2016), NCI-H1092 (obtained in November 2018), and NCI-H2081 (obtained in November 2018) were obtained from ATCC. NCI-H1876, NCI-H1092, and NCI-H2081 cells were maintained in DMEM/F12 media supplemented with HITES [10 nM hydrocortisone (Sigma-Aldrich, #H0135), insulin (0.01 mg/ml), human transferrin (0.0055 mg/ml), sodium selenite (0.005 g/ml) (ITS, Gemini, #400-145), and 10 nM -estradiol (Sigma-Aldrich, #E2257)] and 5% FBS. The cell lines 188 and 97-2 were isolated from genetically engineered SCLC mouse tumors (see below for description of cell line generation) and maintained in RPMI 1640 media supplemented with HITES and 10% FBS. All cells were grown at 37C in the presence of 5% CO2. Fresh aliquots of cells were thawed every 4 to 6 months.

The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 (N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

CDK2 degraders. Synthesis and characterization of the small-molecule CDK2 degraders TMX-2138 and TMX-2172 and the negative degrader ZXH-7035 (structurally similar to the CDK2 binding region of TMX-2138 and TMX-2172 but lacking the cereblon recruiting element) are described previously (25).

293FT cells stably transduced with bicistronic lentiviruses expressing (i) a fusion between DCK* and the POI and (ii) GFP were seeded at a density of 0.25 106 cells/ml in 25 ml of media in a 15-cm dish (Corning, 353025). Two days later, the cells were counted and resuspended in media to a concentration of 10 106 cells/ml. The sample was passed through a mesh strainer (Thermo Fisher Scientific, #352235). The GFP fluorescence of the cells was analyzed by FACS using a Fortessa Aria II instrument. The brightest 1% of cells were collected in an Eppendorff tube, replated in a six-well dish, and expanded. This process was repeated three to four more times to isolate cells expressing the desired GFP levels.

Jurkat cells were first transduced with PLL3.7-Cas9-IRES-Neo. Neomycin-resistant cells with confirmed Cas9 expression were then superinfected with pLX304-ASCL1-DCK*-IRES-GFP or pLX304-DCK*-IRES-GFP, and transduced cells were selected with blasticidin. The blasticidin-resistant cells were then prepared for FACS sorting as above. In total, the brightest 1% of cells were FACS-sorted three times to isolate cells expressing the desired GFP levels. Jurkat cells expressing Cas9 and DCK*-FOXP3 were made in an analogous manner.

Cell pellets were lysed in a modified EBC lysis buffer [50 mM tris-Cl (pH 8.0), 250 mM NaCl, 0.5% NP-40, and 5 mM EDTA] supplemented with a protease inhibitor cocktail (cOmplete, Roche Applied Science, #11836153001). Whole-cell extracts were quantified using the Bradford protein assay. For experiments with 293FT cells, 10 g of protein per sample was boiled after adding 3 sample buffer (6.7% SDS, 33% glycerol, 300 mM dithiothreitol, and bromophenol blue) to a final concentration of 1; resolved by SDSpolyacrylamide gel electrophoresis (PAGE) using either 12.5% SDS-PAGE, Mini-Protean TGX 4 to 15% gels (Bio-Rad, #456-1086), or Criterion TGX gels (Bio-Rad, #5671085); semi-dry transferred onto nitrocellulose membranes; blocked in 5% milk in tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 hour; and probed with the indicated primary antibodies overnight at 4C. Membranes were then washed three times in TBS-T, probed with the indicated horseradish peroxidaseconjugated secondary antibodies for 1 hour at room temperature, and washed three times in TBS-T. Bound antibodies were detected with enhanced chemiluminescence Western blotting detection reagents [Immobilon (Thermo Fisher Scientific, #WBKLS0500) or SuperSignal West Pico (Thermo Fisher Scientific, #PI34078)]. The primary antibodies and dilutions used were as follows: rabbit anti-IKZF1 (Cell Signaling Technology, #5443S) at 1:1000, rabbit anti-V5 (Bethyl Laboratories, #A190-120A) at 1:1000, rabbit anti-DCK (Abcam, #151966) at 1:2000, rabbit anti-ASCL1 (Abcam, #ab211327) at 1:1000, rabbit anti-CDK2 (Cell Signaling Technology, #2546S) at 1:1000, mouse anti-P62 (Abcam, #ab56416) at 1:1000, rabbit antiLC3-I and LC3-II (Cell Signaling Technology, #3868S) at 1:1000, rabbit anti-ATG7L (Cell Signaling Technology, #8558S) at 1:1000, rabbit anti-Beclin1 (Cell Signaling Technology, #3495S) at 1:1000, rabbit anti-FIP200 (Cell Signaling Technology, #12436S) at 1:1000, rabbit -phospho-RB1 S795 (Cell Signaling Technology, #9301P) at 1:1000, mouse -RB1 4H1 (Cell Signaling Technology, #9309S) at 1:1000, mouse anti-actin (Sigma-Aldrich; clone AC-15, #A3854) at 1:25,000, mouse anti-Cas9 (Cell Signaling Technology, #14697) at 1:1000, mouse anti-vinculin (Sigma-Aldrich; #V9131) at 1:10,000, and mouse anti-actin (Cell Signaling Technology, #3700S) at 1:10,000. The secondary antibodies and dilutions used were goat anti-mouse (Pierce) at 1:10,000 and goat anti-rabbit (Pierce) at 1:5000.

A total of 750,000 293FT IKZF1-V5 cells per well were seeded in six-well dishes in a volume of 2 ml. On the next day, drugs to be added were diluted from a 10 mM stock (stored at 20C) into 0.5 ml of media before being added to the cells. The final volume in each well was then made up to 3 ml by adding a second drug in 0.5 ml or adding 0.5 ml of drug-free media.

Myeloma cells were seeded in 10-cm plates at a density of 0.75 106 cells/ml in a total volume of 8 ml. On the next day, the desired drug was diluted from a 10 mM stock (stored at 20C) into 1 ml of media, which was added to the intended well to achieve the desired final concentration. The final volume in each well was then made up to 10 ml by adding a second drug in 1 ml or adding 1 ml of drug-free media. After 24 hours, the cells were harvested for analysis.

293FT cells stably transduced with bicistronic lentiviruses encoding (i) IKZF1, DCK*, or DCK*-IKZF1 (IKZF1 cells, DCK* cells, and DCK*-IKZF1 cells, respectively) and (ii) GFP, as well as corresponding EV control cells, were seeded into six-well plates at 20,000 cells per well in 2.5 ml of media. The next day, 1 M BVdU dissolved in DMSO was diluted into media to prepare 6 stock solutions of BVdU at concentrations of 6 mM, 600 M, 60 M, 6 M, and 600 nM. For each stock solution, DMSO concentration was adjusted to a final concentration of 0.6%. Each well in the six-well dish received 0.5 ml of a 6 stock solution of BVdU to achieve final concentrations of 1 mM, 100 M, 10 M, 1 M, and 100 nM, respectively. A total of 0.5 ml of media with 0.6% DMSO was added to the sixth well as a control. Four days later, the cells were collected and counted using a Vi-Cell XR cell counter.

293FT cells were seeded as above at a density of 20,000 cells per well in 2 ml of media. A stock solution of 10 mM POM in DMSO was diluted into media to prepare a 6 M stock solution of POM. Cells received 0.5 ml of the 6 M POM stock solution to achieve an eventual final concentration of 1 M or 0.5 ml of control media. The next day, BVdU was added as described above, and cell proliferation was analyzed as above.

Jurkat cells expressing Cas9 and either ASCL1, ASCL1-DCK*, or DCK* alone were plated at 0.05 106 cells/ml per well in a 12-well plate and treated with increasing concentrations of BVdU (0, 1, 10, 100, 200, or 500 M). Six days later, the cells were counted using a Vi-Cell XR cell counter. For ASCL1 sgRNA rescue experiments, Jurkat cells expressing Cas9 and ASCL1-DCK* cells were superinfected with pLentiGuide-Purobased lentiviruses expressing sgRNAs targeting ASCL1 or a nontargeting sgRNA (sgCTRL). The cells were selected with puromycin, and expression of ASCL1 was analyzed by immunoblot analysis. The cells were then subjected to the BVdU assay as described above.

293FT cells stably transduced with bicistronic lentiviruses encoding (i) DCK*, DCK*-IKZF1, DCK*-K-RAS (G12V), DCK*-Cyclin D1, DCK*-PAX5, DCK*-FOXP3, and DCK*-MYC and (ii) GFP, as well as corresponding EV control cells, were seeded into 384-well plates (Corning, #3764) at 200 cells per well in 30 l of media. The next day, 1 M BVdU dissolved in DMSO was diluted into media to prepare 4 stock solutions of BVdU at concentrations of 4 mM, 2 mM, 1 mM, 400 M, 200 M, 100 M, 40 M, 20 M, 4 M, and 400 nM. On each plate, 10 l of each stock concentration of BVdU was added to two columns (32 wells) to achieve final concentrations of 1 mM, 500 M, 250 M, 100 M, 50 M, 25 M, 10 M, 5 M, 1 M, and 100 nM. Ten microliters of control media was added to four columns. Four days later, the cells were analyzed using an Acumen laser scanning cytometer (TTP Biosciences). GFP fluorescence was quantified by defining the metric GFP-positive object to identify GFP-positive cells while excluding debris or cell fragments.

Determination of Z. DCK* and DCK*-IKZF1 cells were seeded into 384-well plates (Corning, #3764) in 30 l of media at a density of 200 cells per well and allowed to adhere overnight. For each plate, an HPD300 dispenser (Hewlett-Packard) was used to add 4 nl of POM to a final concentration of 1 M to half the wells. An equal volume of DMSO was added to the other half of the plate. The next day, BVdU was added to the entire plate at a concentration of 10 M for the plate of DCK* cells and 100 M for the plate of DCK*-IKZF1 cells. Four days later, the cells were analyzed using an Acumen laser scanning cytometer (TTP Biosciences). The number of GFP-positive objects in each well was measured, and a Z statistic was calculated comparing the POM-treated wells to the DMSO-treated wells.

High-throughput chemical library screening. DCK* and DCK*-IKZF1 293FT cells were seeded into 384-well plates (Corning, #3764) at a density of 200 cells per well in a volume of 30 l of media. A custom-built Seiko Compound Transfer Robot was used to pin transfer 100 nl per well of small-molecule stock solutions from the wells of a drug library plate to the wells of assay plate, such that each well of the assay plate received a unique small molecule. An HPD300 non-contact dispenser (Hewlett-Packard) was used to dispense 100 nl of POM and dipyridamole into columns 23 and 24 and to add 100 nl of DMSO to columns 1 and 2. The final concentrations of POM and dipyridamole were 10 M and 12.5 M, respectively. The next day, 10 l of BVdU stock solution was added to columns 2 to 24 of each of the DCK-IKZF1 and DCK* assay plates, respectively. The concentration of the BVdU stock solution was calculated to achieve the desired final concentration of BVdU (10 M in DCK* assay plates and 100 M in DCK*-IKZF1 assay plates) in the well.

After 4 days, the GFP fluorescence of each assay plates was quantified using an Acumen scanning laser cytometer. For each plate, the average and SD of the GFP fluorescence of wells in columns 3 to 22 were calculated. For each well on an assay plate, the GFP fluorescence was converted to a z score using the formula: z(well) = [GFP (well) GFP (plate)] / GFP (plate), where GFP (plate) is the mean GFP fluorescence for that plate and GFP (plate) is the SD for that plate.

High-throughput chemical library screening (in-well competition assay). DCK*-IKZF1 (GFP) and DCK* (Td) cells were mixed together in a 1:1 ratio and then seeded into 384-well plates (Corning, #3764) at a density of 400 cells per well in 30 l of media. Pin transfer from IMiD derivative library plates and dispensation of POM and dipyridamole were performed as described above. The next day, 10 l of BVdU stock solution was added to columns 2 to 24 of each plate to achieve a final concentration of 100 M. After 4 days, the GFP and TdTomato fluorescence of each assay plate was quantified using an Acumen scanning laser cytometer. For each well, the ratio of GFP/tdTomato fluorescence was calculated and normalized to the values in the well that received DMSO and BVdU. The resulting values were converted to a heatmap using Morpheus (Broad Institute).

Determination of Z. 293FT IKZF1-Fluc cells were seeded into 96-well plates at a density of 2000 cells per well in a volume of 50 l of media and incubated overnight at 37C. The next day, an additional 50-l media and POM (final concentration of 2 M) was added to 30 wells of the plate (rows B to G, columns 2 to 6). Control media containing DMSO was added to 30 wells of the plate (rows B to G, columns 7 to 11). A Dual-Glo assay (Promega) was performed by first aspirating all media from the tissue culture plates. Twenty-five microliters of a 1:1 dilution of Dual-Glo luciferase assay reagent in phosphate-buffered saline (PBS) was added to wells and incubated for 10 min. Luminescent signal was measured with a plate reader. Stop & Glo reagent (12.5 l) was then added to the wells, incubated for 10 min, and luminescent signal was measured. The average Fluc/Rluc ratio for cells treated with DMSO and POM was calculated, and a Z statistic was calculated.

High-throughput library screening using Fluc/Rluc readout. IKZF1-Fluc assay plates were generated by plating 293FT IKZF1-Fluc cells into 384-well plates. For the 8-hour treatment arm, cells were plated at a density of 4000 cells per well. A custom-built Seiko Compound Transfer Robot was used to pin transfer 100 nl per well of small molecule from the drug library plate to the assay plate, such that each well of the assay plates received a unique small molecule. After 8 hours, the plates were shaken out and blotted on clean paper towels to remove the media. A Thermo Multidrop Combi was used to dispense 20 l of a 1:1 dilution of Dual-Glo luciferase reagent, and the plates were shaken for 10 min. Firefly luciferase signal was quantified using an EnVision plate reader. A Thermo Multidrop Combi was used to dispense 10 l of Dual-Glo Stop + Glo reagent, and the plates were shaken for 10 min. Renilla luciferase signal was quantified using an EnVision plate reader. For each plate, the ratios of the Firefly/Renilla luciferase signals were converted to a Z-distribution as outlined above. For the 4-day treatment arm, the experiment was performed in an analogous manner, but the cells were plated at a density of 200 cells per well and were incubated for 4 days before analysis.

Gene-targeting sgRNAs and appropriate controls were designed using the rule set described at the Genetic Perturbation Program (GPP) portal ( Oligonucleotides were flanked by polymerase chain reaction (PCR) primer sites, and PCR was used to amplify DNA using NEBNext kits. The PCR products were purified using Qiagen PCR cleanup kits and cloned into pXPR_BRD003 using Golden Gate cloning reactions. Pooled libraries were amplified using electrocompetent Stbl4 cells. Viruses were generated as outlined at the GPP portal. The sgRNA library (CP1080, M-AB34) was custom-designed to target cancer-relevant druggable genes. It consisted of 5566 sgRNAs targeting 788 genes (7 sgRNAs targeting each gene) and 300 nontargeting sgRNAs as controls (table S6).

Jurkat cells that had been infected with PLL3.7-Cas9-IRES-Neo and subsequently maintained in G418 were then superinfected with pLX304 ASCL1-DCK*-V5-IRES-GFP or pLX304 DCK*-V5-IRES-GFP and placed under blasticidin selection. Blasticidin-resistant cells were sorted for GFP expression (top 1%) three times by FACS. Protein abundance of ASCL1-DCK* or DCK* alone was confirmed by immunoblot analysis, and functionality of ASCL1-DCK* or DCK* alone was determined using BVdU sensitivity and rescue experiments with sgRNAs targeting ASCL1. Cas9 expression was confirmed by immunoblot analysis, and Cas9 activity was confirmed using a Cas9 GFP reporter [pXPR_011 (Addgene, #59702)] (39) that showed near maximal editing 10 days after infection.

On day 0, ASCL1-DCK* and DCK* cells expressing Cas9 were expanded and then counted. For each line, 2.2 107 cells (4000 cells per sgRNA) were pelleted and resuspended at 2 106 cells/ml in media supplemented with polybrene (8 g/ml) and infected at a multiplicity of infection (MOI) of ~0.3 with the sgRNA druggable library (CP1080, M-AB34) described above. The cells mixed with polybrene and virus were then plated in 1-ml aliquots onto 12-well plates and centrifuged at 434g for 2 hours at 30C. Sixteen hours later (day 1), the cells were collected, pooled, and centrifuged to remove the virus and polybrene, and the cell pellet was resuspended in complete media at 2 105 cells/ml and plated into nontissue culturetreated t175 flasks. The cells were then cultured for 48 hours before being placed under puromycin (1 g/ml) drug selection at 4 105 cells/ml.

A parallel experiment was performed on day 3 to determine the MOI. To do this, the cells infected with the sgRNA library and mock-infected cells were plated at 4 105 cells/ml in the presence or absence of puromycin. After 72 hours (day 6), cells were counted using the Vi-Cell XR cell counter, and the MOI was calculated (which ranged from 0.2 to 0.3 for each replicate) using the following equation: (# of puromycin-resistant cells infected with the sgRNA library / # total cells surviving without puromycin after infection with the sgRNA library) (# of puromycin-resistant mock-infected cells / # total mock-infected cells).

On day 6 after MOI determination, puromycin-resistant cells were pooled, collected, and counted, and 1 108 cells were replated at a concentration of 4 105 cells/ml in complete media containing puromycin (1 g/ml). The remaining cells were discarded. On day 8, again, the puromycin-resistant cells were pooled, collected, and counted, and 1 108 cells were replated at a concentration of 4 105 cells/ml in complete media containing puromycin (1 g/ml).

On day 10, puromycin-resistant cells were pooled, collected, and counted. A total of 2 107 cells were collected and washed in PBS, and the cell pellets were frozen for genomic DNA isolation for the initial time point before BVdU selection. Then, 2 107 cells were resuspended in complete media (now without puromycin) containing either 200 or 500 M BVdU at a final concentration of 5 104 cells/ml and plated into t175-cm flasks. Thus, at least 1000 cells per sgRNA were introduced into BVdU selection.

On day 15, cells treated with 200 or 500 M BVdU were collected and counted. A total of 10 106 cells from each arm of the screen were then resuspended in complete media containing either 200 or 500 M BVdU at a final concentration of 5 104 cells/ml and plated into t175-cm flasks. The remaining cells were centrifuged and washed in PBS, and the cell pellets were frozen. Again, at least 1000 cells per sgRNA were maintained under BVdU selection.

On day 20, cells treated with 200 or 500 M BVdU were collected and counted. A total of 10 106 cells from each arm of the screen were then resuspended in complete media containing either 200 or 500 M BVdU at a final concentration of 5 104 cells/ml and plated into t175-cm flasks. If available, the remaining cells were centrifuged and washed in PBS, and the cell pellets were frozen. Again, at least 1000 cells per sgRNA were maintained under BVdU selection.

On day 25, all remaining cells were collected and counted. The remaining cells were divided in aliquots of 6 106 cells (which corresponds to 1000 cells per sgRNA) and washed in PBS, and the cell pellets were frozen for genomic DNA isolation for the final time point after BVdU selection. The screen was performed in two biological replicates.

Following completion of the screen, genomic DNA was isolated using a Qiagen Genomic DNA midi prep kit (catalog no. 51185) according to the manufacturers protocol. Raw Illumina reads were normalized between samples using log2[(sgRNA reads/total reads for sample) 1 106 + 1]. The initial time point data (day 10) were then subtracted from the end time point after BVdU selection (day 25) to determine the relative enrichment of each individual sgRNA after BVdU treatment using hypergeometric analysis and the STARS algorithm. A q value cutoff of <0.25 was used to call hits. The averaged data from two biological replicates were used for all analyses.

293FT cells stably transduced with bicistronic lentiviruses encoding (i) DCK*-IKZF1 and GFP and (ii) DCK* and TdTomato were mixed together at a ratio of 1:99. Pooled cells were plated at a density of 20,000 cells per well of a six-well plate and in a total volume of 2 ml of media. Cells received 0.5 ml of the 6 M POM stock solution to achieve an eventual final concentration of 1 M or 0.5 ml of control media. The next day, the cells received 0.5 ml of 600 M BVdU stock solution or 0.5 ml of control media. Cells were collected for FACS analysis on days 0, 3, 6, 10, and 14. After each time point, cells were reseeded at 20,000 cells per well and treated with fresh BVdU (or DMSO).

DCK*-IKZF1 293FT cells were infected with a mixture of two lentiviruses encoding Cas9 and either (i) sgDCK and mCherry or (ii) sgCTRL and BFP (blue fluorescent protein). The two lentiviruses were mixed together such that the ratio of mCherry-positive to BFP-positive cells after infection and puromycin selection was 1:99. An analogous experiment was set up using a lentivirus encoding sgCTRL and mCherry. The pool of infected cells was plated at 20,000 cells per well in a six-well plate and then cultured in media containing either 100 M BVdU or DMSO for 21 days. Cells were collected for FACS analysis on days 1, 6, 18, and 33. After each time point, cells were reseeded at 20,000 cells per well and treated with fresh BVdU.

Jurkat cells that had been stably infected to express Cas9 and DCK*-FOXP3 were superinfected with lentivirus encoding either (i) sgDCK and mCherry or (ii) sgCTRL and BFP. These cells were mixed together and analyzed by FACS to achieve a final ratio of mCherry-positive to BFP-positive cells of 1:99. The pool of infected cells was plated at 40,000 cells/ml in a six-well plate and then cultured in media containing either 100 M BVdU or DMSO for 14 days. Cells were collected for FACS analysis on days 6 and 14.

The Jurkat cells expressing Cas9 and ASCL1-DCK* that were used for the CRISPR-Cas9 screen described above were superinfected with lentiviruses encoding sgRNAs targeting CDK2, ASCL1, or a nontargeting sgRNA as a control, the fluorescent protein mCherry and a puromycin resistance gene or with a lentivirus encoding a nontargeting sgRNA as a control, and the fluorescent protein BFP and a puromycin resistance gene (see schema in fig. S16F). The cells were selected with puromycin. mCherry puromycin-resistant cells were then mixed with BFP puromycin-resistant cells at a 1:3 ratio as determined by FACS analysis. The mixed cells were plated at 5 104 cells/ml and then cultured in media containing 500 M BVdU or DMSO (0) for 18 days. FACS analysis was performed every 6 days. After each FACS analysis, fresh BVdU was added, and the density of the cells was adjusted to 5 104 cells/ml with fresh media.

Cells were counted using a Vi-Cell XR cell counter and were plated at a concentration of 4 105 cells/ml per well for NCI-H1092, 188, and 97-2 SCLC cell lines or at 1 106 cells/ml per well for the NCI-H1876 SCLC cell line in six-well plates. Cells were then treated with the CDK2 degraders TMX-2138, TMX-2172, or the negative degrader ZXH-7035 (Neg Deg) at 500 nM for 36 hours for NCI-H1092 and NCI-H1876 human SCLC cell lines or 8 hours for 188 and 97-2 mouse SCLC cell lines. For half-life time determination with cycloheximide, 97-2 cells were treated with CDK2 degraders for 4 hours before the addition of cycloheximide at 150 g/ml. Cells were harvested at the indicated times after addition of cycloheximide.

293FT cells were seeded in six-well plates at a density of 750,000 cells per well in 2.5 ml of media per well. The next day, the cells were treated with the indicated concentrations of Spautin-1, POM, or DMSO for 24 hours. Multiple myeloma cells were seeded in 10-cm plates at a density of 0.75 106 cells/ml in a total volume of 9 ml of media. The next day, the cells were treated with the indicated concentrations of Spautin-1, POM, or DMSO. RNA was extracted using an RNeasy mini kit (Qiagen, #74106) according to the manufacturers instructions. RNA concentration was determined using the NanoDrop 8000 (Thermo Fisher Scientific). cDNA was generated by reverse transcription using the AffinityScript qPCR (quantitative PCR) cDNA Synthesis kit (Agilent, 600559) according to the manufacturers instructions. qPCR was performed using the LightCycler 480 (Roche) with the LightCycler 480 Probes Master Kit (Roche) and TaqMan probes (Thermo Fisher Scientific) according to the manufacturers instructions. The Ct values for each probe were then normalized to the Ct value of ACTB for that sample. The data from each experiment were then normalized to the control to determine the relative fold change in mRNA expression. The following TaqMan probes were used: Hs00958474_m1 (IKZF1 human), ASCL1 human Hs04187546_g1 for detection of endogenous ASCL1, ASCL1 human Hs05000540_s1 for detection of the exogenous ASCL1-DCK* fusion, ACTB human Hs01060665_m1, Ascl1 mouse Mm03058063_m1, and Actb mouse Mm00607939_s1. All quantitative calculations were performed using the 2Ct method using Beta Actin (ACTB) as a reference gene.

For the positive selection small-molecule screen, GFP fluorescence for each well was normalized to untreated wells. For each library drug, normalized GFP fluorescence was plotted as a function of library drug concentration. Each drug treatment was performed in duplicate. Data were analyzed and plotted using GraphPad Prism v6, median inhibitory concentration (IC50) values were determined using the log (inhibitor) versus response -- Variable slope (four parameters) analysis module, and area under the curve (AUC) values were determined using the AUC analysis module (13). For the positive selection CRISPR-Cas9 BVdU resistance screen, the relative fold enrichment of each individual sgRNA after BVdU treatment was calculated using both Broad Institutes hypergeometric analysis and the STARS algorithm to determine a rank list of candidate ASCL1 stabilizer genes ranked by q value, where statistical significance is q < 0.25.

For all other experiments, statistical significance was calculated using unpaired, two-tailed Students t test. P values were considered statistically significant if the P value was <0.05. For all figures, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent SD unless otherwise indicated.

Acknowledgments: We thank the members of the Kaelin and Oser laboratories for helpful discussions. Special thanks to the ICCB (Longwood) at HMS for assistance with small-molecule screens, to W. Gao for generation of adenoviral vectors used for recombination cloning, and D. Hong for generation of SCLC mouse cell lines. Funding: W.G.K. is supported by an NIH R35 grant and is an HHMI investigator. M.G.O. is supported by a Damon Runyon Cancer Research Foundation Clinical Investigator Award and an NCI/NIH KO8 grant (no. K08CA222657). V.K. is supported by an American Society of Hematology Research Training Award and T32 NIH Training Grant CA009172. J.A.P. is funded by an NIGMS grant R01 GM132129. E.S.F. is funded by NCI R01CA2144608. N.S.G. is funded by NIH R01 CA214608-03. M.I. is supported by an Internationalisation Fellowship from the Carlsberg Foundation. C.J.O. is supported by an NIH/NCI Pathway to Independence Award (R00CA190861). Author contributions: V.K., L.D., and B.L.L. performed experiments and, together with W.G.K. and M.G.O., designed experiments, analyzed data, and assembled and wrote the manuscript. J.A.M. helped design experiments. A.C.W. and A.H.S. performed experiments. M.I. designed and synthesized the IMiD library. J.P. and C.J.O. measured CRBN binding and cellular activity of candidate IMiDs; J.B. supervised these experiments. I.S.H. and J.E.E. constructed the Ludwig anticancer and antimetabolite libraries and helped analyze data from the screen. E.D., X.L., and S.J.B. synthesized and characterized Spautin-1 derivatives. J.A.P. performed TMT global proteomic profiling of Spautin-1. S.P.G. supervised these experiments. K.A.D. and E.S.F. analyzed TMT proteomic data. K.J.B. determined the half-lives of luciferase fusion proteins and the Z of the dual-luciferase system. J.G.D. helped analyze data from the CRISPR screen. M.T., T.Z., and N.S.G. helped generate and validate CDK2 degraders. Competing interests: W.G.K. has financial interests in Lilly Pharmaceuticals, Fibrogen, Agios Pharmaceuticals, Cedilla Therapeutics, Nextech Invest, Tango Therapeutics, and Tracon Pharmaceuticals. N.S.G. is a founder, science advisory board member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro, and Soltego (board member). E.S.F. is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Jengu Therapeutics (board member), and Neomorph Inc. E.S.F. is an equity holder of C4 Therapeutics. E.S.F. consults or has consulted for Novartis, AbbVie, Astellas, Deerfield, EcoR1, and Pfizer. The Fischer laboratory receives or has received research funding from Novartis, Deerfield, and Astellas. The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. M.G.O. has sponsored research agreements with Lilly Pharmaceuticals and Takeda Pharmaceuticals. V.K. has consulted for Cedilla Therapeutics. S.J.B. is on the SAB of Adenoid Cystic Carcinoma Foundation. J.B. is an employee, executive, and shareholder of Novartis AG (Basel, Switzerland). J.G.D. consults for Agios, Foghorn Therapeutics, Maze Therapeutics, Merck, and Pfizer; J.G.D. consults for and has equity in Tango Therapeutics. J.G.D.s interests were reviewed and are managed by the Broad Institute in accordance with its conflict of interest policies. I.S.H. is a consultant for ONO Pharmaceuticals (USA). V.K. and W.G.K. are inventors on a patent application on positive selection assays to identify protein degraders, which was filed by the Dana-Farber Cancer Institute (U.S. patent application number 16/332,921, filed on 13 March 2019 and published on 1 August 2019). N.S.G., M.T., and T.Z. are named inventors on patent applications covering Cdlk2 degraders described in the paper, and which were filed by the Dana Farber Cancer Institute (U.S. Provisional Application No. 62/829,302, filed April 4, 2019 and U.S. Provisional Application No: 62/981,334, filed February 25, 2020). 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. Additional data related to this paper may be requested from the authors. All plasmids are available from the authors.

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Targeting oncoproteins with a positive selection assay for protein degraders - Science Advances

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Programming in the pandemic – Perforce: In open source, crowd is a positive –

The Computer Weekly Developer Network examines the impact of Covid-19 (Coronavirus) on the software application development community.

With only a proportion of developers classified as key workers (where their responsibilities perhaps included the operations-side of keeping mission-critical and life-critical systems up and online), the majority of programmers will have been forced to work remotely, often in solitude.

So how have the fallout effects of this played out?

This post comes from Justin Reock in his role as chief evangelist for open source software (OSS) & Application Programming Interface (API) management at Perforce Software.

Reock reflects upon the use of open source platforms, languages and related technologie in general in light of the Covid-19 global crisis and writes as follows

On the whole, I would argue that open source software has been invaluable during the pandemic.

Crowd-sourced software initiatives and hackathons, protein-folding peer-to-peer networks and foundation sponsorship have all been in play throughout the contagion and many of these initiatives continue forwards.

GitHub has shown us that commits held steady or even increased suggesting (if it is fair to measure that in terms of raw commits without considering quality) that developer productivity has held steady or even gone up.

For many developers, having a shared project and sense of community during a very isolating time for humanity has been uplifting and good for their spirits. Its a reminder that coding together is in fact a social activity, no different than any other collaborative and creative endeavour.

Perhaps the biggest impact and fallout from this whole period of experiences (for programmers, operations staff and the wider software engineering community) will be the acceleration of transformation and DevOps initiatives within businesses.

So many have witnessed the resilience of businesses that have already undergone the DevOps transition (and even watched their profits soar) as we moved to online ordering, contactless delivery and more.

The CI/CD part of the DevOps makeover has always been about dealing with constant change.The mantra of releases are hard, so release often embraces the notion that change is difficult, so organisations should make themselves really good at dealing with it. That meant when the pandemic hit, the seams of our global digital twin were tested. Companies that were capable of quickly refactoring to online experiences, digital goods and other conveniences have now become essential to carrying on a reasonable quality of life in the physical world.

It is one thing to expect the unexpected, and it is quite another to design systems that thrive in unexpected conditions.Whatever requisite effort may need to be invested to achieve DevOps maturity in an organisation, the positive impact it can have to business longevity is now indisputable.

However, especially in segments of the industry that are highly collaborative such as gaming, quality and deadlines have suffered drastically and development teams have blamed it squarely on moving to a remote work model.

NOTE: As a software change management specialist, Perforce has a particularly acute proximity with and close understanding of how games programmers work.

Even enabling employees to work from home was a challenge, as the hardware supply chain which we rely on to deliver our webcams, tablets, and laptops and other tech gear suffered major disruptions: so, all in all, there is no question that organisations, including open source communities, which had already taken steps towards transformation and remote work were able to continue operations smoothly, though not completely without impact.

That said, the overall industry picture is not all rosy, with many segments that rely heavily on peer collaboration taking a hit in quality and productivity.

We hope, of course, for brighter future times for all.

Reock: Commit to commit dear developers, you know you want to.

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Programming in the pandemic - Perforce: In open source, crowd is a positive -

Recommendation and review posted by Alexandra Lee Anderson

TYME Granted U.S. Patent Claims Covering Use of TYME-19 to Treat COVID-19 Infections – Business Wire

BEDMINSTER, N.J.--(BUSINESS WIRE)--Tyme Technologies, Inc. (NASDAQ: TYME), an emerging biotechnology company developing cancer metabolism-based therapies (CMBTs), announced that it has received notification that the United States Patent and Trademark Office has granted additional patent claims related to the Companys metabolomic technology platform. The patent, U.S. Patent No. 10,905,698, is directed to methods for treating COVID-19.

Unlike immune therapies that depend upon the structure of the external virus coat of COVID-19 where the therapy directs its attack, we believe TYME-19 is agnostic to this structure and any mutations to the viral coat. Like other TYME agents, TYME-19 affects cellular metabolism. It constrains viral replication after a virus has inserted its genetic blueprint into an infected cell by inhibiting the ability of the virus to use the cells synthetic apparatus to make viral proteins and lipids. As a result, we believe that TYME-19 diminishes the ability of COVID-19 to hijack an infected cell. TYME intends to initiate the appropriate clinical trials to substantiate the safety and efficacy of TYME-19.

TYME-19 is an investigational compound that is not approved in the U.S. for any disease indication.

About TYME-19

TYME-19 is an oral synthetic member of the bile acid family that the Company also uses in its anticancer compound, TYME-18. Because of its expertise in metabolic therapies, the Company was able to identify TYME-19 as a potent, well characterized antiviral bile acid and has performed preclinical experiments establishing effectiveness against COVID-19. Bile acids have primarily been used for liver disease; however, like all steroids, they are messenger molecules that modulate a number of diverse critical cellular regulators. Bile acids modulate lipid and glucose metabolism and can remediate dysregulated protein folding, with potentially therapeutic effects on cardiovascular, neurologic, immune, and other metabolic systems. Some agents in this class also have antiviral properties. In preclinical testing, TYME-19 repeatedly prevented COVID-19 viral replication without attributable cytotoxicity to the treated cells. Previous preclinical research has also shown select bile acids like TYME-19 have had broad antiviral activity.

About Tyme Technologies

Tyme Technologies, Inc., is an emerging biotechnology company developing cancer therapeutics that are intended to be broadly effective across tumor types and have low toxicity profiles. Unlike targeted therapies that attempt to regulate specific mutations within cancer, the Companys therapeutic approach is designed to take advantage of a cancer cells innate metabolic weaknesses to compromise its defenses, leading to cell death through oxidative stress and exposure to the bodys natural immune system.

With the development of TYME-18 and TYME-19, the Company believes that it is also emerging as a leader in the development of bile acids as potential therapies for cancer and COVID-19. For more information, visit Follow us on social media: Facebook, LinkedIn, Twitter, YouTube and Instagram.

Forward-Looking Statements/Disclosure Notice

In addition to historical information, this press release contains forward-looking statements under the Private Securities Litigation Reform Act that involve substantial risks and uncertainties. Such forward-looking statements within this press release include, without limitation, statements regarding our drug candidates (including SM-88 and TYME- 18) and their clinical potential and non-toxic safety profiles, our drug development plans and strategies, ongoing and planned preclinical or clinical trials, including the proposed TYME-19 proof-of-concept study, preliminary data results and the therapeutic design and mechanisms of our drug candidates. The words believes, expects, hopes, may, will, plan, intends, estimates, could, should, would, continue, seeks, anticipates, and similar expressions (including their use in the negative) are intended to identify forward-looking statements. Forward-looking statements can also be identified by discussions of future matters such as: the effect of the novel coronavirus (COVID-19) pandemic and the associated economic downturn and impacts on the Company's ongoing clinical trials and ability to analyze data from those trials; the cost of development and potential commercialization of our lead drug candidate and of other new products; expected releases of interim or final data from our clinical trials; possible collaborations; and the timing, scope, status, objectives and strategy of our ongoing and planned trials; the success of management transitions; and other statements that are not historical. The forward-looking statements contained in this press release are based on managements current expectations and projections which are subject to uncertainty, risks and changes in circumstances that are difficult to predict and many of which are outside of our control. These statements involve known and unknown risks, uncertainties and other factors which may cause the Companys actual results, performance or achievements to be materially different from any historical results and future results, performance or achievements expressed or implied by the forward-looking statements. These risks and uncertainties include but are not limited to: the severity, duration, and economic and operational impact of the COVID-19 pandemic; that the information is of a preliminary nature and may be subject to change; uncertainties inherent in the cost and outcomes of research and development, including the cost and availability of acceptable-quality clinical supply, and in the ability to achieve adequate start and completion dates, as well as uncertainties in clinical trial design and patient enrollment, dropout or discontinuation rates; the possibility of unfavorable study results, including unfavorable new clinical data and additional analyses of existing data; risks associated with early, initial data, including the risk that the final data from any clinical trials may differ from prior or preliminary study data; final results of additional clinical trials that may be different from the preliminary data analysis and may not support further clinical development; that past reported data are not necessarily predictive of future patient or clinical data outcomes; whether and when any applications or other submissions for SM-88 may be filed with regulatory authorities; whether and when regulatory authorities may approve any applications or submissions; decisions by regulatory authorities regarding labeling and other matters that could affect commercial availability of SM-88; the ability of TYME and its collaborators to develop and realize collaborative synergies; competitive developments; and the factors described in the section captioned Risk Factors of TYMEs Annual Report on Form 10-K filed with the U.S. Securities and Exchange Commission on May 22, 2020, as well as subsequent reports we file from time to time with the U.S. Securities and Exchange Commission available at

The information contained in this press release is as of its release date and TYME assumes no obligation to update forward-looking statements contained in this release as a result of future events or developments.

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TYME Granted U.S. Patent Claims Covering Use of TYME-19 to Treat COVID-19 Infections - Business Wire

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Synthetic Biology Startup Acquires AI Platform To Disrupt The Drug Industry – Forbes

Sean McClain, Co-Founder and CEO of AbSci.

There has been a lot of recent attention on the challenges of delivering COVID-19 vaccines. But there are also challenges in making them. For some of the newer options like those from Johnson & Johnson and Oxford-AstraZeneca, the modified cells used in vaccine production are struggling under the scale of demand. But synthetic biology company AbScis recent acquisition of the artificial intelligence platform, Denovium, could help mitigate this type of challenge in the future.

Unlike mRNA vaccines, the Johnson & Johnson/Oxford-AstraZeneca class of vaccines rely on a type of virus called adenovirus which is known to cause colds in chimpanzees. To address COVID-19, the adenovirus is genetically altered to express the SARS-CoV-2 spike protein which is what ultimately triggers the bodys immune response. Like mRNA vaccines, adenovirus-based vaccines train the body to recognize and fight COVID-19, foregoing the need to inject a person with a weakened version of SARS-CoV-2.

But producing enough adenovirus cells has been a challenge. To make vaccine doses, large volumes of altered adenovirus are produced by replicating cells in bioreactors. But, the scale of production can also cause the cells to weaken. This can result in a reduced output of adenovirus copies. So while these new vaccines may represent a breakthrough in adenovirus-based therapeutics, the process also highlights some critical roadblocks.

One major issue is that drug discovery and drug manufacturing are often disconnected from one another. Drug discovery typically starts with screeningthe process of finding a set of compounds out of 100,000 combinations that can best neutralize a targeted weak point of a disease. But when a promising protein is identified, it often turns out to be difficult to scale effectively.

Once a therapeutic compound is identified, researchers must then determine if it works well with a group of similar cells called a cell-line. By inserting the compound into the cellswhich then divide and multiply in a bioreactorthe cells act like factories to produce greater volumes of the compound of choice. But, as in the case with adenovirus-producing cells, not all cells can maintain their functions at large volumes. If the protein compound doesnt work well in a scalable cell-line, researchers often have to go back to the drawing board to find a new compound and start again.

Many in the biopharma space are aware of this inefficient process. The synthetic biology company AbSci has spent years developing a platform solution that streamlines the workflow. [Our platform] is simultaneously a drug discovery and manufacturing platform that allows you to discover your drug and the cell line that can manufacture [it], says AbSci CEO, Sean McClain. Were finally uniting drug discovery and manufacturing the first time.

AbSci refers to their core process as their Protein Printing platform, not because it uses ink and paper to make proteins but as an analogy for ease and speed. The first technology [in our platform] is our SoluPro E. coli strain. It has been highly engineered to be more mammalian-like to be able to produce mammalian-like proteins that E. coli wasn't previously capable of doing, says McClain. AbSci also uses what the company calls a folding solution to precisely tailor how proteins fold and therefore function.

Imad Ajjawi, Co-Founder and CBO of Denovium

To find the most effective protein, AbSci alters its folding solutions to create as many protein varieties as possible, often to the order of 10s of millions. The more protein types available, which AbSci refers to as libraries, the higher the likelihood of success. But this also creates a challenge: so many options, but which to choose?

To address this, AbSci recently acquired artificial intelligence company, Denovium. By integrating Denoviums AI platform, AbSci can improve its data analysis via AI models. From there, the company can take the best candidates and find the most effective cell-line to produce the chosen compounds at scale. McClain explains that traditional drug discovery and manufacturing typically takes years. But AbScis platform can take that timeline down to weeks. Were actually able to manufacture [therapeutics] because the dirty secret in pharma is that so many drugs get shelved because [pharma companies] can't actually manufacture them, says McClain.

For McClain, acquiring Denovium is a big step forward for AbScis discovery process. Its going to change the paradigm. Its really a perfect marriage of both data and AI technology. If you don't have good data feeding into your AI model, it's worthless. But if you don't have an AI technology, you can't mine [the data] and get all the benefits, says McClain.

Denoviums co-founder and CBO, Imad Ajjawi, also sees the new collaboration as a significant opportunity. It's really exciting to be a part of AbSci because they have all the data, billions of points that the deep learning engine can now analyze, says Ajjawi. AbScis acquisition also comes on the heels of the companys $65 million Series E in late 2020.

Upgrading the union of biology and AI is important for advancing synthetic biology innovation. But the true potential beneficiaries of this advanced discovery platform are those in need of novel drug options.

AbScis main goal as a company is to bring therapeutics to market more quickly. This technology's impact on healthcare is profound because more drugs and biologics can now enter patients' hands faster, says McClain.

McClain believes that AbScis technology will help speed the process of clinically testing new medications. Faster clinical trial turnarounds could increase the number of drugs approved to address a range of diseases. This could be most impactful for patients with rare or difficult to treat conditions as drug discovery is often prioritized based on how long it takes to find a scalable cell-line.

But though AbSci is working to accelerate drug discovery, the process still takes time. Right now, we have six drugs that are in preclinical or clinical trials. And one of them is actually in phase three. So we could have an improved product here in the next couple of years, says McClain.

As Absci and Denovium finalize their technology integrations, McClain is also looking ahead to build as many partnerships as possible. The more partnerships we do, the more patients were able to affect that at the end of the day, says McClain.

In line with that goal, AbSci today announced a continuation of its partnership with Astellas and Xyphos. AbSci will take on screening and identifying an optimal cell-line for a leading variant of Xyphos MicAbody, a bispecific antibody-like adaptor molecule used in the company's immuno-oncology program.

McClain expects more partnership announcements will follow in the first quarter of 2021. We have some really exciting partnerships that are going to be coming out over this next quarter that I think speak to the [range] of the types of disease states we're working on and the breadth of how the technology can be used within biopharma, says McClain.

Im the founder of SynBioBeta, and some of the companies that I write about are sponsors of the SynBioBeta conference and weekly digest, including AbSci. Thank you to Fiona Mischel and Vinit Parekh for additional research and reporting in this article.

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Synthetic Biology Startup Acquires AI Platform To Disrupt The Drug Industry - Forbes

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