Search

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.

Gene therapy is a promising method for the treatment of vascular disease; however, successful strategies depend on the development of safe and effective delivery technologies with specific targeting to a diseased point of vasculature. Reactive oxygen species (ROS) are overproduced by vascular smooth muscle cells (VSMCs) at critical stages of atherosclerosis progression. Therefore, ROS were exploited as a stimulus for vascular targeted gene delivery in this study. A combination of bio-conjugation methods and controlled reverse addition-fragmentation chain-trasfer (RAFT) polymerization was utilized to synthesize a new ROS-cleavable, pH-responsive mPEG113-b-CP5K-b-PDMAEMA42-b-P(DMAEMA22-co-BMA40-co-PAA24) (PPDDBP) polymer as a nanocarrier for plasmid DNA (pDNA) delivery. The ros degradability of PPDDBP polymers was confirmed by SIN-1-mediated cleavage of CP5K peptide linkers through a shift in GPC chromatogram with an appearance of mPEG shoulder peak and an increase in zeta potential (ζ). The polyplex nanocarrier also demonstrated effective PDNA loading, serum stability, and hemocompatibility, indicating its excellent performance under physiological conditions. The polyplexes demonstrated ideal pH responsiveness for endosomal escape and effective ROS responsiveness for improved targeting in an in vitro model of pathogenic VSMCs in terms of both uptake and expression of reporter gene. These data suggest this novel nanocarrier polyplex system is a promising gene delivery tool for preventing or treating areas of high ROS, such as atherosclerotic lesions.

The recent genome editing revolution has been fueled by the discovery and adaptation of highly specific endonucleases including meganucleases, zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas9. These genome editing technologies permit user-defined genome modifications by creating double-strand DNA breaks and exploiting endogenous DNA repair pathways to introduce DNA sequence changes. Genome editing has entered multiple clinical trials in a range of diseases including HIV, cancer, and hemophilia, and several preclinical successes have been reported for treating models of neuromuscular diseases, including Duchenne muscular dystrophy (DMD). These studies include correction of numerous different mutations in patient-derived muscle cells and stem cells by a variety of genome editing strategies and endonuclease technologies. Preclinical studies have also shown efficacy of genome editing by restoring dystrophin protein expression and improving skeletal muscle physiology in animal models of DMD. This preclinical work highlights the potential for DNA repair therapy to treat DMD and other debilitating and fatal genetic diseases. Ongoing work seeks to address remaining issues including efficient delivery, addressing potential immune response or off-target interactions, and characterizing long-term safety and efficacy.

Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1–6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study. In the mdx mouse model of Duchenne muscular dystrophy, single intravenous administration of AAV-CRISPR–Cas9 vectors provides efficient genome editing and restoration of dystrophin expression lasting for one year.

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications., Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.

MicroRNA (miRNA) are noncoding RNA that provide an endogenous negative feedback mechanism for translation of messenger RNA (mRNA) into protein. Single miRNAs can regulate hundreds of mRNAs, enabling miRNAs to orchestrate robust biological responses by simultaneously impacting multiple gene networks. MiRNAs can act as master regulators of normal and pathological tissue development, homeostasis, and repair, which has recently motivated expanding efforts toward development of technologies for therapeutically modulating miRNA activity for regenerative medicine and tissue engineering applications. This review highlights the tools currently available for miRNA inhibition and their recent therapeutic applications for improving tissue repair.

A new microparticle-based delivery system was synthesized from reactive oxygen species (ROS)-responsive poly(propylene sulfide) (PPS) and tested for “on demand” antioxidant therapy. PPS is hydrophobic but undergoes a phase change to become hydrophilic upon oxidation and thus provides a useful platform for ROS-demanded drug release. This platform was tested for delivery of the promising anti-inflammatory and antioxidant therapeutic molecule curcumin, which is currently limited in use in its free form due to poor pharmacokinetic properties. PPS microspheres efficiently encapsulated curcumin through oil-in-water emulsion and provided sustained, on demand release that was modulated in vitro by hydrogen peroxide concentration. The cytocompatible, curcumin-loaded microspheres preferentially targeted and scavenged intracellular ROS in activated macrophages, reduced in vitro cell death in the presence of cytotoxic levels of ROS, and decreased tissue-level ROS in vivo in the diabetic mouse hind limb ischemia model of peripheral arterial disease. Interestingly, due to the ROS scavenging behavior of PPS, the blank microparticles also showed inherent therapeutic properties that were synergistic with the effects of curcumin in these assays. Functionally, local delivery of curcumin-PPS microspheres accelerated recovery from hind limb ischemia in diabetic mice, as demonstrated using non-invasive imaging techniques. This work demonstrates the potential for PPS microspheres as a generalizable vehicle for ROS-demanded drug release and establishes the utility of this platform for improving local curcumin bioavailability for treatment of chronic inflammatory diseases.

For many neuromuscular disorders, including Duchenne muscular dystrophy, spinal muscular atrophy and myotonic dystrophy, the genetic causes are well known. Gene therapy holds promise for the treatment of these monogenic neuromuscular diseases, and many such therapies have made substantial strides toward clinical translation. Recently, genome engineering tools, including targeted gene editing and gene regulation, have become available to correct the underlying genetic mutations that cause these diseases. In particular, meganucleases, zinc finger nucleases, TALENs, and the CRISPR-Cas9 system have been harnessed to make targeted and specific modifications to the genome. However, for most gene therapy applications, including genome engineering, gene delivery remains the primary hurdle to clinical translation. In preclinical models, genome engineering tools have been delivered via gene-modified cells or by non-viral or viral vectors to correct a diverse array of genetic diseases. In light of the positive results of these studies, genome engineering therapies are being enthusiastically explored for several genetic neuromuscular disorders. This Review summarizes the genome engineering strategies that are currently under preclinical evaluation for the treatment of degenerative neuromuscular disorders, with a focus on the molecular tools that show the greatest potential for clinical translation of these therapies.

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers., Site-specific recombination and CRISPR-Cas9 have been used to generate genetically engineered mouse models of cancer. Here the authors compare sarcomas generated using both systems and see similar genetic and cellular phenotypes, suggesting CRISPR-Cas9 can be used to rapidly generate sarcoma models.

Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy. Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))–pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.

A system has been engineered for temporally controlled delivery of siRNA from biodegradable tissue regenerative scaffolds. Therapeutic application of this approach to silence prolyl hydroxylase domain 2 promoted expression of pro-angiogenic genes controlled by HIF1α and enhanced scaffold vascularization in vivo. This technology provides a new standard for efficient and controllable gene silencing to modulate host response within regenerative biomaterials.

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)], PEG-(DMAEMA-co-BMA), was reversible addition-fragmentation chain transfer (RAFT) synthesized with 0-75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer "50 B") in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape. In vitro delivery of siRNA with polymer 50 B produced up to 94% protein-level knockdown of the model gene luciferase. The PEG corona of the NPs blocked nonspecific interactions with constituents of human whole blood, and the relative hydrophobicity of polymer 50 B increased NP stability in the presence of human serum or the polyanion heparin. When injected intravenously, 50 B NPs enhanced blood circulation half-life 3-fold relative to more standard PEG-DMAEMA (0 B) NPs (p0.05), due to improved stability and a reduced rate of renal clearance. The 50 B NPs enhanced siRNA biodistribution to the liver and other organs and significantly increased gene silencing in the liver, kidneys, and spleen relative to the benchmark polymer 0 B (p0.05). These collective findings validate the functional significance of tuning the balance of cationic and hydrophobic content of polyplex NPs utilized for systemic siRNA delivery in vivo.

Macrophages represent an important therapeutic target, because their activity has been implicated in the progression of debilitating diseases such as cancer and atherosclerosis. In this work, we designed and characterized pH-responsive polymeric micelles that were mannosylated using “click” chemistry to achieve CD206 (mannose receptor)-targeted siRNA delivery. CD206 is primarily expressed on macrophages and dendritic cells and upregulated in tumor-associated macrophages, a potentially useful target for cancer therapy. The mannosylated nanoparticles improved the delivery of siRNA into primary macrophages by 4-fold relative to the delivery of a nontargeted version of the same carrier (p < 0.01). Further, treatment for 24 h with the mannose-targeted siRNA carriers achieved 87 ± 10% knockdown of a model gene in primary macrophages, a cell type that is typically difficult to transfect. Finally, these nanoparticles were also avidly recognized and internalized by human macrophages and facilitated the delivery of...

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mousemodel of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force.This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD., Muscular Dystrophy Association (Award MDA277360), National Institutes of Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant R01DK097768)

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for cancer research, and recently the CRISPR/Cas9 system has been increasingly utilized to model cancer in mice. Here, we used CRISPR/Cas9 to generate two primary mouse models of sarcoma, undifferentiated pleomorphic sarcoma (UPS) in a GEMM, and malignant peripheral nerve sheath tumor (MPNST) in wild-type mice, to demonstrate the versatility of the system to generate multiple soft-tissue sarcoma subtypes. Because CRISPR technology is becoming more prevalent in cancer modeling, it is critical to thoroughly evaluate if these models are indeed comparable as tools to study cancer biology compared to conventional GEMMs initiated by recombinase technology. We used two Kras-driven sarcoma models of UPS generated with either Cre recombinase technology or CRISPR/Cas9 technology and compared the mutational profiles, histology, and growth kinetics of these models. KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ (KC) mice received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft-tissue sarcomas. Compared to the Cre/loxP model, we determined that sarcomas generated with CRISPR/Cas9 had similar growth kinetics, histology, copy number variation, and mutational load as assessed by whole-exome sequencing. We also demonstrated that off-target mutations in the sarcomas initiated by the Cas9 endonuclease were rare in tumors. Finally, we analyzed the Cas9-mediated indels present in tumors as genetic barcodes, which will enable future studies of tumor heterogeneity and clonality. These results show that sarcomas generated with CRISPR/Cas9 technology are similar to sarcomas generated with conventional modeling techniques. Ultimately this work corroborates CRISPR/Cas9-generated mouse models with traditional GEMMs phenotypically and genotypically, and expands the range of sarcoma mouse models available for research. Citation Format: Jianguo Huang, Mark Chen, Melodi Javid Whitley, Hsuan-Cheng Kuo, Eric S. Xu, Andrea Walens, Yvonne M. Mowery, David Van Mater, William C. Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Omar M. Lopez, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Anupama Reddy, Sandeep S. Dave, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A17.

The discovery of RNAi in the late 1990s unlocked a new realm of therapeutic possibilities by enabling potent and specific silencing of theoretically any desired genetic target. Better elucidation of the mechanism of action, the impact of chemical modifications that stabilize and reduce nonspecific effects of siRNA molecules, and the key design considerations for effective delivery systems has spurred progress toward developing clinically-successful siRNA therapies. A logical aim for initial siRNA translation is local therapies, as delivering siRNA directly to its site of action helps to ensure that a sufficient dose reaches the target tissue, lessens the potential for off-target side effects, and circumvents the substantial systemic delivery barriers. While locally injected or topically applied siRNA has progressed into numerous clinical trials, an enormous opportunity exists to develop sustained-release, local delivery systems that enable both spatial and temporal control of gene silencing. This review focuses on material platforms that establish both localized and controlled gene silencing, with emphasis on the systems that show most promise for clinical translation.

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for pre-clinical studies, but this approach is costly and time-consuming. Here, we show that the CRISPR/Cas9 system can be used to efficiently complement existing GEMMs of sarcoma and generate primary sarcomas in wild type mice. Mice with the genotype KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft tissue sarcomas. These tumors arose with kinetics similar to those generated using the Cre-loxP system to activate oncogenic Kras and delete Trp53 alleles. Additionally, we injected an adenovirus containing Cas9 and sgRNAs targeting Nf1 and Trp53 into the sciatic nerve of wild type mice. These mice formed malignant peripheral nerve sheath tumors (MPNSTs) in the same timeframe as MPNSTs generated using the Cre-loxP system to delete Nf1 and Ink4a/Arf alleles in GEMMs. These data demonstrate that CRISPR/Cas9 can be used to generate soft tissue sarcomas in wild type mice. Moreover, these results suggest that this technology can complement existing GEMMs for rapid assessment of tumor-modifying genes. These tools should decrease the time and expense associated with employing autochthonous mouse models of sarcoma for preclinical research. Citation Format: Jianguo Huang, Mark Chen, Melodi J. Whitley, Hsuan-Cheng Kuo, Andrea Walens, Yvonne M. Mowery, David V. Mater, William Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Using CRISPR/Cas9 to generate primary soft tissue sarcoma in genetically engineered and wild-type mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2017-2810

Peripheral arterial disease is often modeled by surgical induction of hind limb ischemia (HLI) in mice to study collateral vessel development. However, there is a need for methodologies that provide intravital, multifunctional, quantitative data on ischemic recovery for robust evaluation of new therapeutics. Here, we apply hyperspectral imaging and optical coherence tomography (OCT) to longitudinally assess hemoglobin oxygen saturation (SaO 2 ) and vessel morphology in response to a novel therapy. Injectable microspheres loaded with curcumin were synthesized from reactive oxygen species (ROS)-responsive poly(propylene) sulfide (PPS) to provide “on demand”, local release of the antioxidant drug curcumin to reduce tissue-damaging oxidative stress in a mouse model of diabetic HLI. Curcumin-PPS microspheres significantly reduced intracellular ROS in LPS-activated RAW 264.7 macrophages and rescued viability of 3T3 fibroblasts treated with cytotoxic levels of H 2 O 2 in vitro . HLI was induced in FVB mice with streptozotocin-induced diabetes, and curcumin-PPS or blank-PPS microspheres were injected into the ischemic limb. Curcumin-PPS significantly improved recovery of SaO 2 in the ischemic footpad relative to blank-PPS and vehicle (saline) groups over a one week time course evaluated with hyperspectral imaging (n≥8/group, p

A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity.

Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceutical inhibitors, but technologies that enable robust, tissue-specific intracellular delivery must be developed before effective clinical translation can be achieved. A pH-responsive, smart polymeric nanoparticle (SPN) with matrix metalloproteinase (MMP)-7-dependent proximity-activated targeting (PAT) is described here. The PAT-SPN was designed to trigger cellular uptake and cytosolic delivery of siRNA once activated by MMP-7, an enzyme whose overexpression is a hallmark of cancer initiation and progression. The PAT-SPN is composed of a corona-forming PEG block, an MMP-7-cleavable peptide, a cationic siRNA-condensing block, and a pH-responsive, endosomolytic terpolymer block that drives self-assembly and forms the PAT-SPN core. With this novel design, the PEG corona shields cellular interactions until it is cleaved in MMP-7-rich environments, shifting SPNζ-potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase in carrier internalization. The PAT-SPN exhibited pH-dependent membrane disruptive behavior that enabled siRNA escape from endo-lysosomal pathways. Efficient intracellular siRNA delivery and knockdown of the model enzyme luciferase in R221A-Luc mammary tumor cellssignificantly depended on MMP-7 pre-activation. These combined data indicate that the PAT-SPN provides a promising new platform for tissue-specific, proximity-activated siRNA delivery to MMP-rich pathological environments.

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼ 80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100 D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50 D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50 D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50 D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.

Peripheral arterial disease (PAD) is an atherosclerotic disease of the extremities that leads to high rates of myocardial infarction and stroke, increased mortality, and reduced quality of life. PAD is especially prevalent in diabetic patients, and is commonly modeled by hind limb ischemia in mice to study collateral vessel development and test novel therapies. Current techniques used to assess recovery cannot obtain quantitative, physiological data non-invasively. Here, we have applied hyperspectral imaging and swept source optical coherence tomography (OCT) to study longitudinal changes in blood oxygenation and vascular morphology, respectively, intravitally in the diabetic mouse hind limb ischemia model. Additionally, recommended ranges for controlling physiological variability in blood oxygenation with respect to respiration rate and body core temperature were determined from a control animal experiment. In the longitudinal study with diabetic mice, hyperspectral imaging data revealed the dynamics of blood oxygenation recovery distally in the ischemic footpad. In diabetic mice, there is an early increase in oxygenation that is not sustained in the long term. Quantitative analysis of vascular morphology obtained from Hessian-filtered speckle variance OCT volumes revealed temporal dynamics in vascular density, total vessel length, and vessel diameter distribution in the adductor muscle of the ischemic limb. The combination of hyperspectral imaging and speckle variance OCT enabled acquisition of novel functional and morphological endpoints from individual animals, and provides a more robust platform for future preclinical evaluations of novel therapies for PAD.

Duchenne muscular dystrophy (DMD) is a monogenic and fatal genetic disorder characterized by muscle wasting, loss of ambulation, and death by the age of 20 due to the loss of functional dystrophin – an essential musculoskeletal protein. Gene therapy has held promise for treating monogenetic diseases, however, delivery barriers and the requirements for costly repetitive treatments are a few of the challenges of technologies in the therapeutic pipeline. Recently, the CRISPR/Cas9 system has been adapted for genome editing to make specific modifications in the host genome. This technology would enable permanent genome corrections and a potentially curative approach to DMD.Previously we adapted CRISPR/Cas9 for targeted deletions of regions of the human dystrophin gene that restore expression of a functional protein (Ousterout et al., Nature Communications (in press)). The delivery of Cas9 with single guide RNAs (sgRNAs) targeting the introns surrounding exons 51 and 45-55 created double strand breaks which are repaired through non-homologous end joining to create genomic deletions of these regions. These deletions restored the reading frame and recovered dystrophin expression in cultured patient-derived myoblasts. Therapeutically, this approach could be used to convert the fatal DMD phenotype to the milder phenotype associated with partial dystrophin function characteristic of Becker muscular dystrophy.Here we utilize adeno-associated virus (AAV) to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD. AAV vectors were designed to package and deliver genes encoding the Staphylococcus aureus Cas9 (SaCas9), due to its smaller size (3.2 kb) compared to the widely used Cas9 from Streptococcus pyogenes. Two single guide RNAs (sgRNAs) were designed to target sites flanking exon 23 of the dystrophin gene and validated to direct SaCas9-mediated deletion of this exon, which contains a premature stop codon in this model. Mice were anesthetized and AAV1 containing the SaCas9 and sgRNA expression cassettes was injected into the gastrocnemius muscle of 8 week old mice. At 4 and 8 weeks post-injection, the gastrocnemius was harvested and genomic DNA and mRNA were extracted. PCR of the genomic DNA demonstrated the expected ~1200 bp deletion of exon 23. Further, RT-PCR of the extracted mRNA showed removal of the 216 bp encoding exon 23. Furthermore, sgRNAs compatible with SaCas9 have been developed and validated for the deletion of human exon 51.This work demonstrates proof-of-principle of CRISPR/Cas9-mediated genome editing in skeletal muscle in an adult mammal, opening up new possibilities for gene therapy and the study of gene function. Optimization of delivery and activity is underway to improve the therapeutic potential of this approach. Additional work to characterize functional improvement in the mouse hind limb is also ongoing. This work establishes CRISPR/Cas9-mediated genome editing as a potential therapeutic approach to DMD.

We have recently developed a platform for sustained, local release of siRNA loaded endosomolytic nanoparticles (siNPs) from biodegradable poly(ester urethane) (PEUR) tissue engineering scaffolds [1]; this platform was designed to achieve local, gene-specific silencing to promote healing of tissue defects. We are developing this platform to promote local angiogenesis through sustained silencing of prolyl hydroxylase domain protein 2 (PHD2) [2], a negative regulator of the hypoxia induced factor 1α (HIF1α).

Aberrant overexpression of proinflammatory molecules is believed to be a key mediator in the formation of chronic skin wounds, and the inhibition of these signals may be an effective therapeutic strategy to promote healing. Small interfering RNA (siRNA) can provide gene-specific silencing and may present a safe and effective route for knockdown of inflammatory or other target proteins in chronic skin wounds.siRNA suffers from delivery barriersThe siRNA delivery barriers have been overcome using a nanoparticulate carrier that protects siRNA and responds to pH gradients in the endo-lysosomal pathway to mediate cytosolic delivery. Nanoparticle incorporation into a biodegradable PUR scaffold provides a means for controlling the delivery kinetics of siRNA-loaded carriers. Furthermore, the PUR is injectable, making it feasible for clinical use, and provides a porous tissue template for cell in-growth during tissue regeneration and remodeling. This local siRNA delivery platform can be tuned to optimize release kinetics for specific pathologies.siRNA may provide a new class of biologic drugs that will outperform growth factor approaches, which have shown only moderate clinical success. The new platform presented here may provide clinicians with an improved option for wound care.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Injectable, “smart” microspheres that are sensitive to both temperature and pH have been fabricated and tested for controlled delivery of therapeutic proteins to ischemic skeletal muscle. A library of copolymers composed of N-isopropyl acrylamide (NIPAAm), propyl acrylic acid (PAA), and butyl acrylate (BA) was used to fabricate microspheres using a double emulsion method, and an optimal formulation made from copolymers composed of 57 mol% NIPAAm, 18 mol% PAA, and 25 mol% BA copolymers was identified. At 37°C and pH representative of ischemic muscle (i.e., pH 5.2–7.2), these microspheres produced sustained, diffusion-controlled release, and at normal, physiologic pH (i.e. pH 7.4), they underwent dissolution and rapid clearance. Delivery of fibroblast growth factor 2 (FGF-2) was used to confirm that protein bioactivity was retained following microsphere encapsulation/release based on a dose-dependent increase in NIH3T3 fibroblast proliferation in vitro. Microsphere-loaded or free Cy5.5-labeled albumin was injected into ischemic and control gastrocnemii of mice following unilateral induction of hind limb ischemia to model peripheral arterial disease. In the ischemic limb at day 3.5 and day 7, there was higher local retention of the protein delivered via microspheres relative to injected free protein (p

Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease characterized by progressive muscle wasting, loss of ambulation, and typically death in the third decade of life due to respiratory and cardiac complications. DMD results from deleterious mutations in the dystrophin gene that disrupts the translational reading frame and causes a complete lack of dystrophin protein. Becker muscular dystrophy (BMD) is similar to DMD in that it is the result of deletions in the dystrophin gene. However these deletions maintain the translational reading frame and result in the production of an internally truncated but partially functional dystrophin protein. The BMD phenotype is typically less severe than DMD, and thus converting DMD to a BMD-like phenotype by restoring the dystrophin reading frame is a widely explored therapeutic strategy. CRISPR/Cas9 can target precise loci to make specific DNA sequence changes in the genome. We have previously used S. pyogenes Cas9 (SpCas9) to restore dystrophin expression in immortalized myoblasts from DMD patients by deleting dystrophin exon 51 to repair the disrupted reading frame. A promising therapeutic application of this strategy involves in vivo viral delivery of the CRISPR/Cas9 system by AAV, however AAV cannot efficiently package the large SpCas9 gene along with full size promoters. Therefore we have made use of AAV delivery of the smaller S. aureus Cas9 (SaCas9) to delete exon 23 in the mouse dystrophin gene in vivo in the mdx mouse model of DMD and showed restored dystrophin expression and functional recovery. Here we are continuing this work by preparing a SaCas9 system targeted to the human dystrophin gene. gRNAs were designed to target the intronic regions flanking exon 51 and tested in vitro in HEK293T cells as well as immortalized DMD patient myoblasts that are correctable by removal of exon 51. The expected deletion was confirmed by PCR and sequencing of the genomic DNA and dystrophin cDNA. Western blot of lysates from differentiated cells confirmed restoration of dystrophin protein expression. We have also demonstrated exon 51 deletion in vivo following AAV delivery of this CRISPR/Cas9 system to the muscles of a mouse model containing the full length human dystrophin gene. Ongoing work involves testing this approach in a novel dystrophic mouse model carrying a mutated version of the human dystrophin gene that is correctable by exon 51 deletion. This work is important to the continued development of a translational strategy for gene editing as a potentially curative treatment for DMD.

Duchenne muscular dystrophy (DMD) is a highly prevalent genetic disorder leading to muscle wasting, loss of ambulation, and premature death by the third decade of life. DMD is caused by gene deletions, duplications, or nonsense mutations leading to the loss of dystrophin, an essential musculoskeletal protein. Gene therapy has held tremendous promise for the treatment of monogenic disorders, yet an effective gene replacement therapy has been elusive. Genome editing has been established as a potential approach to correct the dystrophin gene in cultured human cells by excising non-essential exons from the dystrophin gene producing a shortened yet in-frame dystrophin protein (1). In contrast to gene replacement therapy, genome editing repairs the causative mutation in the native genomic context with the potential for permanent gene repair. Recently, we and others have demonstrated that CRISPR/Cas9 genome editing in neonatal and adult mouse models of DMD restores dystrophin expression, improves muscle biochemistry, and strengthens muscle force generation (2-4). However, further optimization of the approach for systemic gene correction is still needed.To target the dystrophin gene in the mdx mouse, the 3.2kb S. aureus Cas9 and two guide RNAs (gRNA) targeting intronic regions surrounding exon 23 were packaged into an adeno-associated virus (AAV). Double stranded breaks created by Cas9 were repaired with the relatively efficient non-homologous end joining pathway leading to excision of the nonsense mutation in exon 23. AAV vectors were injected intramuscularly into the tibialis anterior muscle in adult mice and intravenously into neonatal mice and adult mice and characterized for gene deletions, dystrophin restoration, and improvements in muscle physiology.Local correction restored overall dystrophin levels to 8% by western blot with 67% of muscle fibers positive for dystrophin by immunofluorescence. Repeated cycles of eccentric contraction showed 60% resistance to damage compared to sham-treated mice. Systemic correction was achieved through IP injection into P2 neonates with dystrophin restoration primarily in the cardiac muscle and skeletal muscle surrounding the peritoneal cavity. IV administration in adult mice restored dystrophin expression in the cardiac muscle. Improved systemic distribution and correction was achieved with intravenous administration into P2 neonates with AAV8 or AAV9 (Fig. 1Fig. 1).Figure 1Intravenously administered AAV restores dystrophin in cardiac and skeletal muscle in P2 neonates after 8 weeks of treatement. a) Cardiac muscle. b) Tibialis anterior muscle. Scale bar = 200µm, green - dystrophin, blue - DAPI.View Large Image | Download PowerPoint SlideThis study establishes CRISPR/Cas9-based gene editing as a promising approach for the treatment of DMD. Ongoing work to improve the efficiency and safety of in vivo gene editing includes the incorporation of muscle specific promoters, minimization of vector packaging, and optimization of AAV serotype.1. Ousterout et al. Nat Comm 2015. | 2. Nelson et al. Science 2015 | 3. Tabebordbar et al. Science 2015 | 4. Long et al. Science 2015

A new micelle drug carrier that consists of a diblock polymer of propylene sulfide (PS) and N,N-dimethylacrylamide (poly(PS₇₄-b-DMA₃₁₀)) has been synthesized and characterized for site-specific release of hydrophobic drugs to sites of inflammation. Propylene sulfide was first polymerized using a thioacyl group transfer (TAGT) method with the RAFT chain transfer agent (CTA) 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (CEP), and the resultant poly(PS₇₄-CEP) macro-CTA was used to polymerize a second polymer block of DMA using reversible addition-fragmentation chain transfer (RAFT). The formation of the poly(PS₇₄-b-DMA₃₁₀) diblock polymer was confirmed by ¹H NMR spectra and gel permeation chromatography (GPC). Poly(PS₇₄-b-DMA₃₁₀) formed 100 nm micelles in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Micelles loaded with the model drugs Nile red and DiO were used to demonstrate the ROS-dependent drug release mechanism of these micelles following treatment with hydrogen peroxide (H₂O₂), 3-morpholinosydnonimine (SIN-1), and peroxynitrite. These oxidants were found to oxidize the micelle PPS core, making it more hydrophilic and triggering micelle disassembly and cargo release. Delivery of poly(PS₇₄-b-DMA₃₁₀) micelles dual-loaded with the Forster Resonance Energy Transfer (FRET) fluorophore pair DiI and DiO was used to prove that endogenous oxidants generated by lipopolysaccharide (LPS)-treated RAW 264.7 macrophages significantly increased release of nanocarrier contents relative to macrophages that were not activated. In vitro studies also demonstrated that the poly(PS₇₄-b-DMA₃₁₀) micelles were cytocompatible across a broad range of concentrations. These combined data suggest that the poly(PS₇₄-b-DMA₃₁₀) micelles synthesized using a combination of TAGT and RAFT have significant potential for site-specific drug delivery to tissues with high levels of oxidative stress.

The recent elucidation of molecular regulators of apoptosis and their roles in cellular oncogenesis has motivated the development of biomacromolecular anticancer therapeutics that can activate intracellular apoptotic signaling pathways. Pharmaceutical scientists have employed a variety of classes of biologics toward this goal, including antisense oligodeoxynucleotides, small interfering RNA, proteins, antibodies, and peptides. However, stability in the in vivo environment, tumor-specific biodistribution, cell internalization, and localization to the intracellular microenvironment where the targeted molecule is localized pose significant challenges that limit the ability to directly apply intracellular-acting, pro-apoptotic biologics for therapeutic use. Thus, approaches to improve the pharmaceutical properties of therapeutic biomacromolecules are of great significance and have included chemically modifying the bioactive molecule itself or formulation with auxiliary compounds. Recently, promising advances in delivery of pro-apoptotic biomacromolecular agents have been made using tools such as peptide "stapling", cell penetrating peptides, fusogenic peptides, liposomes, nanoparticles, smart polymers, and synergistic combinations of these components. This review will discuss the molecular mediators of cellular apoptosis, the respective mechanisms by which these mediators are dysregulated in cellular oncogenesis, the history and development of both nucleic-acid and amino-acid based drugs, and techniques to achieve intracellular delivery of these biologics. Finally, recent applications where pro-apoptotic functionality has been achieved through delivery of intracellular-acting biomacromolecular drugs will be highlighted.

Antibody–drug conjugate therapy entails targeted killing of cancer cells with cytotoxic compounds covalently linked to tumor-specific antibodies and shows promise in the treatment of several human cancers. Current antibody–drug conjugate designs that incorporate a disulfide linker between the antibody and cytotoxic drug are inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing. It is presumed that antigen-dependent endocytosis leads to disulfide linker reduction and intracellular release of free drug, but direct demonstration of such a mechanism is lacking. To determine whether the disulfideN-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker would be reduced during endocytic recycling of the anti-HER2 antibody trastuzumab (Herceptin, Genentech), we synthesized a trastuzumab-SPP-Rhodamine red conjugate and developed a linker cleavage assay by using the self-quenching property of this fluorophore. In breast carcinoma SKBr3 cells, no SPP linker cleavage was observed, as detected by fluorescence dequenching upon internalization. By contrast, the conjugate did display fluorescence dequenching when diverted to the lysosomal pathway by geldanamycin, an effect partly due to proteolytic degradation rather than disulfide reduction. To understand why linker reduction was inefficient, we measured redox potentials of endocytic compartments by expressing a redox-sensitive variant of GFP fused to various endocytic proteins. Unexpectedly, we found that recycling endosomes, late endosomes, and lysosomes are not reducing, but oxidizing and comparable with conditions in the endoplasmic reticulum. These results suggest that intracellular reduction is unlikely to account for the potency of disulfide-linked antibody–drug conjugates.