The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Targeted Enzyme Activity Imaging with Quantitative Phase Microscopy.

Quantitative phase imaging (QPI) is a powerful optical imaging modality for label-free, rapid, and three-dimensional (3D) monitoring of cells and tissues. However, molecular imaging of important intracellular biomolecules such as enzymes remains a largely unexplored area for QPI. Herein, we introduce a fundamentally new approach by designing QPI contrast agents that allow sensitive detection of intracellular biomolecules. We report a new class of bio-orthogonal QPI-nanoprobes for in situ high-contrast refractive index (RI) imaging of enzyme activity. The nanoprobes feature silica nanoparticles (SiO2 NPs) having higher RI than endogenous cellular components and surface-anchored cyanobenzothiazole-cysteine (CBT-Cys) conjugated enzyme-responsive peptide sequences. The nanoprobes specifically aggregated in cells with target enzyme activity, increasing intracellular RI and enabling precise visualization of intracellular enzyme activity. We envision that this general design of QPI-nanoprobes could open doors for spatial-temporal mapping of enzyme activity with direct implications for disease diagnosis and evaluating the therapeutic efficacy.

In Vivo Cellular Magnetic Imaging: Labeled vs. Unlabeled Cells.

Superparamagnetic iron oxide (SPIO)-labeling of cells has been applied for magnetic resonance imaging (MRI) cell tracking for over 30 years, having resulted in a dozen or so clinical trials. SPIO nanoparticles are biodegradable and can be broken down into elemental iron, and hence the tolerance of cells to magnetic labeling has been overall high. Over the years, however, single reports have accumulated demonstrating that the proliferation, migration, adhesion and differentiation of magnetically labeled cells may differ from unlabeled cells, with inhibition of chondrocytic differentiation of labeled human mesenchymal stem cells (hMSCs) as a notable example. This historical perspective provides an overview of some of the drawbacks that can be encountered with magnetic labeling. Now that magnetic particle imaging (MPI) cell tracking is emerging as a new in vivo cellular imaging modality, there has been a renaissance in the formulation of SPIO nanoparticles this time optimized for MPI. Lessons learned from the occasional past pitfalls encountered with SPIO-labeling of cells for MRI may expedite possible future clinical translation of (combined) MRI/MPI cell tracking.

Opportunities for Molecular Imaging in Multiple Sclerosis Management: Linking Probe to Treatment.

Imaging has been a critical component of multiple sclerosis (MS) management for nearly 40 years. The visual information derived from structural MRI, that is, signs of blood-brain barrier disruption, inflammation and demyelination, and brain and spinal cord atrophy, are the primary metrics used to evaluate therapeutic efficacy in MS. The development of targeted imaging probes has expanded our ability to evaluate and monitor MS and its therapies at the molecular level. Most molecular imaging probes evaluated for MS applications are small molecules initially developed for PET, nearly half of which are derived from U.S. Food and Drug Administration-approved drugs and those currently undergoing clinical trials. Superparamagnetic and fluorinated particles have been used for tracking circulating immune cells (in situ labeling) and immunosuppressive or remyelinating therapeutic stem cells (ex vivo labeling) clinically using proton (hydrogen 1 [1H]) and preclinically using fluorine 19 (19F) MRI. Translocator protein PET and 1H MR spectroscopy have been demonstrated to complement imaging metrics from structural (gadolinium-enhanced) MRI in nine and six trials for MS disease-modifying therapies, respectively. Still, despite multiple demonstrations of the utility of molecular imaging probes to evaluate the target location and to elucidate the mechanisms of disease-modifying therapies for MS applications, their use has been sparse in both preclinical and clinical settings.

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs. METHODS AND RESULTS: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine. CONCLUSIONS: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Furin-Mediated Self-Assembly of Olsalazine Nanoparticles for Targeted Raman Imaging of Tumors.

Olsalazine (Olsa) is a broad-spectrum anti-cancer agent acting as a DNA-methylation inhibitor. When conjugated to 2-cyano-6-aminobenzothiazole and a peptide substrate specific for the tumor-overexpressed enzyme furin, it can self-assemble into nanoparticles that can be detected by chemical-exchange saturation-transfer magnetic-resonance imaging (CEST MRI). We report here that these nano-assemblies can also be detected with high specificity in furin-overexpressing tumor cells by Raman spectroscopy with a distinct scattering signature and demonstrate the utility of this sensing mechanism in vitro and in vivo. Our findings suggest that Raman spectroscopy could be used for high-resolution image-guided surgery to precisely delineate tumor margins during and after resection in real-time as well as to determine microscopic tumor invasion and multifocal locoregional tumor spread, which are currently impossible to visualize with available imaging technologies, including CEST MRI.

Monitoring diffuse injury during disease progression in experimental autoimmune encephalomyelitis with on resonance variable delay multiple pulse (onVDMP) CEST MRI.

Multiple sclerosis (MS) is an autoimmune disorder that targets myelin proteins and results in extensive damage in the central nervous system in the form of focal lesions as well as diffuse molecular changes. Lesions are currently detected using T1-weighted, T2-weighted, and gadolinium-enhanced magnetic resonance imaging (MRI); however, monitoring such lesions has been shown to be a poor predictor of disease progression. Chemical exchange saturation transfer (CEST) MRI is sensitive to many of the biomolecules in the central nervous system altered in MS that cannot be detected using conventional MRI. We monitored disease progression in an experimental autoimmune encephalomyelitis (EAE) model of MS using on resonance variable delay multiple pulse (onVDMP) CEST MRI. Alterations in onVDMP signal were observed in regions responsible for hindlimb function throughout the central nervous system. Histological analysis revealed glial activation in areas highlighted in onVDMP CEST MRI. onVDMP signal changes in the 3rd ventricle preceded paralysis onset that could not be observed with conventional MRI techniques. Hence, the onVDMP CEST MRI signal has potential as a novel imaging biomarker and predictor of disease progression in MS.

Furin-mediated intracellular self-assembly of olsalazine nanoparticles for enhanced magnetic resonance imaging and tumour therapy.

Among the strategies used for enhancement of tumour retention of imaging agents or anticancer drugs is the rational design of probes that undergo a tumour-specific enzymatic reaction preventing them from being pumped out of the cell. Here, the anticancer agent olsalazine (Olsa) was conjugated to the cell-penetrating peptide RVRR. Taking advantage of a biologically compatible condensation reaction, single Olsa-RVRR molecules were self-assembled into large intracellular nanoparticles by the tumour-associated enzyme furin. Both Olsa-RVRR and Olsa nanoparticles were readily detected with chemical exchange saturation transfer magnetic resonance imaging by virtue of exchangeable Olsa hydroxyl protons. In vivo studies using HCT116 and LoVo murine xenografts showed that the OlsaCEST signal and anti-tumour therapeutic effect were 6.5- and 5.2-fold increased, respectively, compared to Olsa without RVRR, with an excellent 'theranostic correlation' (R2 = 0.97) between the imaging signal and therapeutic response (normalized tumour size). This furin-targeted, magnetic resonance imaging-detectable platform has potential for imaging tumour aggressiveness, drug accumulation and therapeutic response.

In Vivo Imaging of Composite Hydrogel Scaffold Degradation Using CEST MRI and Two-Color NIR Imaging.

Hydrogel scaffolding of stem cells is a promising strategy to overcome initial cell loss and manipulate cell function post-transplantation. Matrix degradation is a requirement for downstream cell differentiation and functional tissue integration, which determines therapeutic outcome. Therefore, monitoring of hydrogel degradation is essential for scaffolded cell replacement therapies. We show here that chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can be used as a label-free imaging platform for monitoring the degradation of crosslinked hydrogels containing gelatin (Gel) and hyaluronic acid (HA), of which the stiffness can be fine-tuned by varying the ratio of the Gel:HA. By labeling Gel and HA with two different NIR dyes having distinct emission excitation frequencies, we show here that the HA signal remains stable for 42 days, while the Gel signal gradually decreases to <25% of its initial value at this time point. Both imaging modalities were in excellent agreement for both the time course and relative value of CEST MRI and NIR signals (R2=0.94). These findings support the further use of CEST MRI for monitoring biodegradation and optimizing of gelatin-containing hydrogels in a label-free manner.

Development of Zinc-Specific iCEST MRI as an Imaging Biomarker for Prostate Cancer.

The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, in vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using 19 F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF-BAPTA as a fluorinated Zn-binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10-fold difference in contrast following glucose-stimulated zinc secretion in vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. In vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.

Carbon Dots as a New Class of Diamagnetic Chemical Exchange Saturation Transfer (diaCEST) MRI Contrast Agents.

While carbon dots (C-dots) have been extensively investigated pertaining to their fluorescent, phosphorescent, electrochemiluminescent, optoelectronic, and catalytic features, their inherent chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) properties are unknown. By virtue of their hydrophilicity and abundant exchangeable protons of hydroxyl, amine, and amide anchored on the surface, we report here that C-dots can be adapted as effective diamagnetic CEST (diaCEST) MRI contrast agents. As a proof-of-concept demonstration, human glioma cells were labeled with liposomes with or without encapsulated C-dots and implanted in mouse brain. In vivo CEST MRI was able to clearly differentiate labeled cells from non-labeled cells. The present findings may encourage new applications of C-dots for in vivo imaging in deep tissues, which is currently not possible using conventional fluorescent (near-infrared) C-dots.

Migratory potential of transplanted glial progenitors as critical factor for successful translation of glia replacement therapy: The gap between mice and men.

Neurological disorders are a major threat to public health. Stem cell-based regenerative medicine is now a promising experimental paradigm for its treatment, as shown in pre-clinical animal studies. Initial attempts have been on the replacement of neuronal cells only, but glial progenitors (GPs) are now becoming strong alternative cellular therapeutic candidates to replace oligodendrocytes and astrocytes as knowledge accumulates about their important emerging role in various disease processes. There are many examples of successful therapeutic outcomes for transplanted GPs in small animal models, but clinical translation has proved to be challenging due to the 1,000-fold larger volume of the human brain compared to mice. Human GPs transplanted into the mouse brain migrate extensively and can induce global cell replacement, but a similar extent of migration in the human brain would only allow for local rather than global cell replacement. We review here the mechanisms that govern cell migration, which could potentially be exploited to enhance the migratory properties of GPs through cell engineering pre-transplantation. We furthermore discuss the (dis)advantages of the various cell delivery routes that are available, with particular emphasis on intra-arterial injection as the most suitable route for achieving global cell distribution in the larger brain. Now that therapeutic success has proven to be feasible in small animal models, future efforts will need to be directed to enhance global cell delivery and migration to make bench-to-bedside translation a reality.

Clinical Tracking of Cell Transfer and Cell Transplantation: Trials and Tribulations.

Cell therapy has provided unprecedented opportunities for tissue repair and cancer therapy. Imaging tools for in vivo tracking of therapeutic cells have entered the clinic to evaluate therapeutic cell delivery and retention in patients. Thus far, clinical cell tracking studies have been a mere proof of principle of the feasibility of cell detection. This review centers around the main clinical queries associated with cell therapy: Have cells been delivered correctly at the targeted site of injection? Are cells still alive, and, if so, how many? Are cells being rejected by the host, and, if so, how severe is the immune response? For stem cell therapeutics, have cells differentiated into downstream cell lineages? Is there cell proliferation including tumor formation? At present, clinical cell tracking trials have only provided information on immediate cell delivery and short-term cell retention. The next big question is if these cell tracking tools can improve the clinical management of the patients and, if so, by how much, for how many, and for whom; in addition, it must be determined whether tracking therapeutic cells in every patient is needed. To become clinically relevant, it must now be demonstrated how cell tracking techniques can inform patient treatment and affect clinical outcomes.

Quantification and tracking of genetically engineered dendritic cells for studying immunotherapy.

PURPOSE: Genetically encoded reporters can assist in visualizing biological processes in live organisms and have been proposed for longitudinal and noninvasive tracking of therapeutic cells in deep tissue. Cells can be labeled in situ or ex vivo and followed in live subjects over time. Nevertheless, a major challenge for reporter systems is to identify the cell population that actually expresses an active reporter. METHODS: We have used a nucleoside analog, pyrrolo-2'-deoxycytidine, as an imaging probe for the putative reporter gene, Drosophila melanogaster 2'-deoxynucleoside kinase. Bioengineered cells were imaged in vivo in animal models of brain tumor and immunotherapy using chemical exchange saturation transfer MRI. The number of transduced cells was quantified by flow cytometry based on the optical properties of the probe. RESULTS: We performed a comparative analysis of six different cell lines and demonstrate utility in a mouse model of immunotherapy. The proposed technology can be used to quantify the number of labeled cells in a given region, and moreover is sensitive enough to detect less than 10,000 cells. CONCLUSION: This unique technology that enables efficient selection of labeled cells followed by in vivo monitoring with both optical and MRI. Magn Reson Med 79:1010-1019, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Characterization of tumor vascular permeability using natural dextrans and CEST MRI.

PURPOSE: To investigate the use of natural dextrans as nano-sized chemical exchange saturation transfer (CEST) MRI probes for characterizing size-dependent tumor vascular permeability. METHODS: Dextrans of different molecular weight (10, 70, 150, and 2000 kD) were characterized for their CEST contrast. Mice (N = 5) bearing CT26 subcutaneous colon tumors were injected intravenously with 10 kD (D10, 6 nm) and 70 kD (D70, 12 nm) dextran at a dose of 375 mg/kg. The CEST-MRI signal in the tumors was assessed before and approximately 40 min after each injection using a dynamic CEST imaging scheme. RESULTS: All dextrans of different molecular weights have a strong CEST signal with an apparent maximum of approximately 0.9 ppm. The detectability and effects of pH and saturation conditions (B1 and Tsat ) were investigated. When applied to CT26 tumors, the injection of D10 could produce a significant "dexCEST" enhancement in the majority of the tumor area, whereas the injection of D70 only resulted in an increase in the tumor periphery. Quantitative analysis revealed the differential permeability of CT26 tumors to different size particles, which was validated by fluorescence imaging and immunohistochemistry. CONCLUSIONS: As a first application, we used 10- and 70-kD dextrans to visualize the spatially variable, size-dependent permeability in the tumor, indicating that nano-sized dextrans can be used for characterizing tumor vascular permeability with dexCEST MRI and, potentially, for developing dextran-based theranostic drug delivery systems. Magn Reson Med 79:1001-1009, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

In Vivo Micro-CT Imaging of Human Mesenchymal Stem Cells Labeled with Gold-Poly-L-Lysine Nanocomplexes.

Developing in vivo cell tracking is an important prerequisite for further development of cell-based therapy. So far, few computed tomography (CT) cell tracking studies have been described due to its notoriously low sensitivity and lack of efficient labeling protocols. We present a simple method to render human mesenchymal stem cells (hMSCs) sufficiently radiopaque by complexing 40 nm citrate-stabilized gold nanoparticles (AuNPs) with poly-L-lysine (PLL) and rhodamine B isothiocyanate (RITC). AuNP-PLL-RITC labeling did not affect cellular viability, proliferation, or downstream cell differentiation into adipocytes and osteocytes. Labeled hMSCs could be clearly visualized in vitro and in vivo with a micro-CT scanner, with a detection limit of approximately 2×104 cells/µl in vivo. Calculated HU values were 2.27 /pg of intracellular Au as measured with inductively coupled plasma mass spectrophotometry (ICP-MS), and were linear over a wide range of cell concentrations. This linear CT attenuation was observed for both naked AuNPs and those that were taken up by hMSCs, indicating that the number of labeled cells can be quantified similar to the use of radioactive or fluorine tracers. This approach for CT cell tracking may find applications in CT image-guided interventions and fluoroscopic procedures commonly used for the injection of cellular therapeutics.

Science to Practice: Can MR Imaging Cell Tracking of Macrophage Infiltration Be Used as a Predictive Imaging Biomarker for Transplanted Stem Cell Rejection?

Ferumoxytol-enhanced magnetic resonance (MR) imaging of donor-matched and mismatched stem cell transplants demonstrated decreased signal intensity not only for a xenogeneic mismatch in species but, surprisingly, also for a syngeneic mismatch in sex. MR imaging findings were corroborated with intravital fluorescence microscopy (IVM), where nearly 90% of all ferumoxytol-containing cells were found to be macrophages. Hence, MR imaging cell tracking of infiltrating macrophages may have predictive value in determining whether transplanted stem cell rejection will occur.

In Vivo 19F MR Imaging Cell Tracking of Inflammatory Macrophages and Site-specific Development of Colitis-associated Dysplasia.

Purpose To investigate whether the magnitude of in vivo fluorine 19 (19F) magnetic resonance (MR) imaging signal is associated with subsequent development of colitis-associated dysplasia after in situ fluorination of inflammatory macrophages in a mouse model of inflammatory bowel disease (IBD). Materials and Methods Experiments were approved by the institutional animal care and use committee. Mice in the experimental group (n = 10) were administered azoxymethane and dextran sulfate sodium to induce colitis-associated dysplasia. Five mice were in the noninduced control group. Animals were injected with a commercially available perfluorocarbon and were examined in vivo with an 11.7-T MR imager for up to 110 days. Colons were then harvested followed by high-spatial-resolution ex vivo MR imaging. Multiple colon segments with or without 19F signal were histologically graded and were correlated with 19F signal intensity by using a Spearman correlation test. The signal intensity in mice with colitis-associated dysplasia was compared with that in control mice with a two-tailed Mann-Whitney U test. Results Patchy distributions of 19F signal intensity in the colon wall were seen on in vivo and ex vivo images, representing chronic inflammation as shown by immunohistochemistry. Histologic scores of inflammation and site-specific development of colitis-associated dysplasia in the descending colon showed good correlation with normalized 19F signal intensity (r = 0.88, P = .033 for the ascending colon; r = 0.82, P = .006 for the descending colon). A significantly (P = .002) higher normalized 19F signal-to-noise ratio was found at sites that developed dysplasia (mean, 0.58 ± 0.09 [standard deviation]) as compared with sites that did not (mean, 0.17 ± 0.22). Conclusion 19F MR imaging cell tracking of macrophages can be used to assess local inflammation in a mouse model of IBD. The resulting local 19F signal intensity, representing the magnitude of inflammation, has a positive correlation with the development of colitis-associated dysplasia. © RSNA, 2016 Online supplemental material is available for this article.

Concise Review: Using Stem Cells to Prevent the Progression of Myopia-A Concept.

The prevalence of myopia has increased in modern society due to the educational load of children. This condition is growing rapidly, especially in Asian countries where it has already reached a pandemic level. Typically, the younger the child's age at the onset of myopia, the more rapidly the condition will progress and the greater the likelihood that it will develop the known sight-threatening complications of high myopia. This rise in incidence of severe myopia has contributed to an increased frequency of eye diseases in adulthood, which often complicate therapeutic procedures. Currently, no treatment is available to prevent myopia progression. Stem cell therapy can potentially address two components of myopia. Regardless of the exact etiology, myopia is always associated with scleral weakness. In this context, a strategy aimed at scleral reinforcement by transplanting connective tissue-supportive mesenchymal stem cells is an attractive approach that could yield effective and universal therapy. Sunlight exposure appears to have a protective effect against myopia. It is postulated that this effect is mediated via local ocular production of dopamine. With a variety of dopamine-producing cells already available for the treatment of Parkinson's disease, stem cells engineered for dopamine production could be used for the treatment of myopia. In this review, we further explore these concepts and present evidence from the literature to support the use of stem cell therapy for the treatment of myopia.