Jeff Bulte - US grants

[unreadable] DESCRIPTION (provided by applicant):Stem cells have the potential to treat and possibly cure a variety of disorders, particularly those of the central nervous system (CNS). To develop successful clinical therapies, the fate of these cells after grafting must be monitored in a noninvasive manner. Currently, there is a need to develop in vivo means to track transplanted cells to determine their survival and migration patterns. By attaching a magnetic tag to stem cells, we propose to follow their biodistribution in vivo using MR imaging. We hypothesize that MR monitoring of the extent of cell migration, including the local distribution within the CNS, will allow for optimization of stem cell transplantation protocols, and that the same strategy may eventually be pursued in humans. Specifically, we will label bone marrow stem cells and neural stem cells obtained from mouse embryonic stem (ES) cell lines. Our first aim is to optimize the magnetic labeling procedure using superparamagnetic iron oxides that are coated with a transfection agent. Following magnetic tagging, we will then graft labeled cells into the CNS or inject cells systemically into the following four animal models of neurodegenerative disease: a) Long Evans shaker rat model of dysmyelination (aim 2); b) Twitcher mouse model of globoid cell leukodystrophy (aim 3); c) Sindbis virus rat model of lower motor neuron disease (aim 4); and d) mouse model of Parkinson's disease (aim 5). These models are believed to represent human CNS diseases that are likely candidates for future stem cell therapy. Using serial MR imaging of the same animal over time, unique information on the spatial temporal dynamics of cell migration can be obtained. Specifically, we aim to correlate the obtained MR contrast images with conventional histopathologic labeling and staining techniques for each differentiated cell type, to determine the extent of new myelination or dopaminergic neuron formation and the potential improvement in animal behavior. Upon the completion of our studies, we expect to demonstrate that MR tracking of magnetically labeled stem cells is a valid new technology for studying stem cell based therapies in the CNS, setting the stage for applying this technique in a clinical setting.

DESCRIPTION (provided by applicant): The transplantation or transfusion of therapeutic stem cells has been pursued as a very active research area over the last decade and a remarkable progress has been obtained in animal disease models. To further develop cell-based therapies into the clinic, noninvasive cellular imaging techniques are warranted. These imaging techniques are needed to provide detailed information on the biokinetics of administered cells, cell-tissue interactions including preferred pathways of migration, and cell survival. Several image modalities now fulfill the requirement of being able to noninvasively and repetitively image targeted cells and cellular processes in living organisms. Among these are single photon emission computed tomography (SPECT) and positron emission tomography or PET, which both use radioactive labels, and bioluminescence imaging and MR imaging. When these four modalities are compared, only MR imaging offers near-cellular resolution, with the ability of detecting only a few cells following their labeling with an MR contrast agent. In the last 2-3 years, there has been an enormous growth in the number of publications on MRI cell tracking of stem cells, predominantly in the central nervous system (CNS). It appears appropriate and timely to organize a workshop related to this young research field. We are therefore organizing a workshop entitled "In vivo MR tracking of stem cell transplants in the CNS". The workshop will be held in Madison, Wl, on June 26, 2005 during the 36th Annual Meeting of The American Society for Neurochemistry (ASN) that runs from June 25- 29. This meeting was chosen because of the excellent scientific program and large number of expected attendees. Moreover, several other concurrent ASN workshops are being organized that focus on basic neural stem cell biology, with many internationally renowned speakers, and the proposed MR imaging workshop would thus reach the desired target audience. The specific aim of this proposal is to obtain financial support for travel and lodging costs for 5 speakers, 4 of whom are coming from overseas. It is anticipated that the workshop format will allow an optimal interaction and exchange of information between neuroscientists active in the stem cell field. It will provide them with new research opportunities that are in line with current NIH roadmaps (stem cells, molecular imaging, nanotechnology). The outcome of the workshop will be published in Experimental Neurology and available for the scientific community at large.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance to the development of novel cell therapies using stem cells and progenitors. Compared to other imaging modalities (i.e., PET, SPECT, and bioluminescent imaging), MR imaging has the highest spatial resolution and can provide both anatomical and functional information, but it suffers from a severely high signal-to noise threshold for the detection of cells using suitable labels/tracers. To a certain degree, this limitation has been resolved using intracellular endosomal tagging with super-paramagnetic nanoparticles. However, the magnetic susceptibility-based T2 (*) contrast induced this way has significant drawbacks, including the creation of hypointense "black holes" (obscuring tissue morphology), difficult differentiation between live and dead cells, the presence of hypointense imaging artifacts, uncertainty about long-term metal toxicity, and, most important, dilution of label following cell proliferation. We are proposing a new approach for the MR detection of labeled cells using a CEST (Chemical Exchange Saturation Transfer) reporter gene. The method is based on the expression of amide-enriched artificial proteins, i.e., lysine-rich protein (LRP) and argenine-rich protein (ARP) that can be detected by CEST imaging in the nanomolar range. The advantages of using these molecular probes are: 1) the gene product can be visualized directly without the need of a substrate (no tissue penetration needed); 2) detection sensitivity is not limited by cell proliferation; 3) only live cells should provide CEST contrast; 4) the contrast can be "switched-on" and "switched-off" repeatedly; and 5) double- or triple-cell labeling strategies may be pursued. We have initial data showing that a CEST reporter gene can be cloned, expressed in transfected cells, and specifically detected by MR CEST imaging in phantoms, without affecting cell viability or proliferation. We hypothesize that this detection is also possible in vivo. To achieve this goal, our aim is to synthesize novel, more efficient CEST reporter genes, and to detect double-labeled LRP/ARP transfected glioma cells and neural stem cells individually in live animals. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance to the development of novel cell therapies using stem cells and progenitors. Compared to other imaging modalities (i.e., PET, SPECT, and bioluminescent imaging), MR imaging has the highest spatial resolution and can provide both anatomical and functional information, but it suffers from a severely high signal-to noise threshold for the detection of cells using suitable labels/tracers. To a certain degree, this limitation has been resolved using intracellular endosomal tagging with super-paramagnetic nanoparticles. However, the magnetic susceptibility-based T2 (*) contrast induced this way has significant drawbacks, including the creation of hypointense "black holes" (obscuring tissue morphology), difficult differentiation between live and dead cells, the presence of hypointense imaging artifacts, uncertainty about long-term metal toxicity, and, most important, dilution of label following cell proliferation. We are proposing a new approach for the MR detection of labeled cells using a CEST (Chemical Exchange Saturation Transfer) reporter gene. The method is based on the expression of amide-enriched artificial proteins, i.e., lysine-rich protein (LRP) and argenine-rich protein (ARP) that can be detected by CEST imaging in the nanomolar range. The advantages of using these molecular probes are: 1) the gene product can be visualized directly without the need of a substrate (no tissue penetration needed); 2) detection sensitivity is not limited by cell proliferation; 3) only live cells should provide CEST contrast; 4) the contrast can be "switched-on" and "switched-off" repeatedly; and 5) double- or triple-cell labeling strategies may be pursued. We have initial data showing that a CEST reporter gene can be cloned, expressed in transfected cells, and specifically detected by MR CEST imaging in phantoms, without affecting cell viability or proliferation. We hypothesize that this detection is also possible in vivo. To achieve this goal, our aim is to synthesize novel, more efficient CEST reporter genes, and to detect double-labeled LRP/ARP transfected glioma cells and neural stem cells individually in live animals. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] For patients with Type I diabetes mellitus, islet transplantation provides a moment-to-moment fine regulation of insulin that is unachievable by exogenous insulin injection. Islet transplantation has been further improved by using alginate microcapsules for immunoprotection. However, the outcome of multi-institutional trials has shown that insulin-independence success rates vary widely. Thus, in order to better understand the fate of transplanted islets and the relationship among transplanted islet mass, graft function, and overall glucose homeostasis, an accurate and reproducible method of imaging islets in vivo is required. Rather than conventional direct labeling of cells with an magnetic resonance (MR) contrast agent, we propose to label alginate capsules with a clinically approved superparamagnetic iron oxide (SPIO) formulation-allowing for sensitive MR detectability at a single capsule level. Our preliminary data demonstrate that the in vitro viability and insulin secretory response of murine beta-cells and human islets are unaltered by encapsulation with alginate-SPIO as compared to naked islets over a 6-week period. These "magnetocapsules" (MCs) are also impermeable to proteins greater than 75 kD, enabling immunoprotection of cells by preventing antibody penetration. Furthermore, MC islet cells can restore normoglycemia in vivo in streptozotocin (STZ)-induced diabetic mice for at least 2 months. [unreadable] Before initiating human clinical feasibility studies, we propose to apply our transplanted MC islet approach in a large animal model that closely resembles the human condition using clinical MR scanners and imaging parameters with dedicated injection catheters and imaging guidewires. First we will further optimize our MC preparations and test their functionality and MR properties in vivo using diabetic mice (Aims 1 and 2). We will then assess our immunoprotected and MR-trackable islets for functionality in a STZ-induced diabetic swine model (Aim 3). Due to their strong magnetic properties, the MCs enable the use of MR-compatible catheters for image-guided targeted portal vein injections in real time. We hypothesize that the rate of success of insulin-dependence will depend on successful islet delivery and liver engraftment, as well as persistence of capsule integrity, as monitored by MR imaging. As the MCs are composed of clinical grade and FDA-approved materials, this MR-guided approach will be readily translatable to human diabetic clinical trials for further improvements of islet cell transplantation. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance for the development of novel therapies using stem cells. There is little question that the field of cellular therapeutics will eventually become important to treat, or possibly cure, a variety of neurodegenerative diseases. Thus, further development of sensitive, non-invasive imaging techniques that can be applied clinically is clearly warranted. We propose to develop magnetic particle imaging (MPI) as a novel technique for imaging of stem cells. This imaging modality is based on the non-linear magnetization curve of small superparamagnetic tracers already used with MRI cell tracking, but the technique itself is not related to MRI. In principle, MPI has several advantages over MRI: a) a 1000-fold higher sensitivity; b) the ability to absolutely quantify the amount of magnetic tracers and cells; c) "hot spot" interpretation without the confounding endogenous background signal present in MRI; and d) the absence of obscuring contrast from hemorrhage or traumatic injury. We have preliminary data that magnetically labeled cells can be detected in vitro at low concentrations with a prototype MPI instrument, and that there is a straightforward quantification of cell number and iron content. Following the optimization of labeling procedures with different particles, MPI will be further investigated in vivo in a rat model of focal transient ischemia using middle cerebral artery occlusion. Different doses of mesenchymal and neural stem cells will be infused either intra-arterially or intravenously. The evolution of ischemic stroke will be monitored with various MRI techniques, including perfusion imaging, diffusion weighted imaging, and pH imaging. The therapeutic efficacy of administered stem cells will be assessed using a four-tier behavioral scoring system. The total amount of targeted and localized cells in the ischemic lesion, as determined by MPI, will be correlated with lesion volume and behavioral scores. While this proposal may be viewed as high risk, we believe that all necessary components are in place in order to successfully develop cellular MPI for stem cell therapy of stroke. By using MPI to obtain a better insight in the dynamic processes that govern the in vivo (selective) homing and (non-selective) trapping of cells in the brain and other tissues, we aim to optimize the source of stem cells, administration route, and dose of cell injection, which may ultimately enhance the therapeutic efficacy of stem cell treatment in stroke patients. PUBLIC HEALTH RELEVANCE: Stem cells have the potential to ameliorate or perhaps even cure neurodegenerative diseases such as stroke. We aim to develop a new imaging technique that uses magnetic particles for visualizing transplanted stem cells in the brain. If this new technique is developed successfully, it will help us to understand much better where the cells exactly go into the brain and elsewhere in the body, which will facilitate introducing these stem cells into patients. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In order to develop stem and progenitor cell therapies for treating CNS diseases, it is important to be able to determine the fate of transplanted cells non-invasively over time, including the location, survival, and status of downstream cell differentiation. We propose to use two complementary modalities, magnetic resonance imaging (MRI) and bioluminescence imaging (BLI), to follow the fate (sites of injection, movements, survival, and differentiation) of transplanted cells in two complementary models of motor neuron disease (MND), i.e., a ricin-induced model as a localized, monophasic and a neurotropic Sindbis virus (NSV)-induced as a global, inflammatory disease model. Magnetically labeled and dual luciferase (Luc-BLI)-transducted glial restrictor precursors (GRPs) or embryonic stem cell-derived motor neurons (ESC-MNs) will be transplanted in the spinal cord of rats following the induction of MND disease. We hypothesize that ESC-MNs can improve functional recovery through formation of new motor neurons, while GRPs can neuroprotect host MNs and/or support transplanted ESC-MNs. In order to assess functional recovery, we will perform behavioral analyses, electrophysiologic measurements (motor-evoked potentials), and muscle mass measures, and will determine whether intraparenchymal or intrathecal injections are the optimal route of transplantation. We will use MRI to monitor the accuracy of cell injections, the extent of cell migration and intraparenchymal tissue distribution (white vs. gray matter), whereas dual luciferase reporter BLI will be used to report on the survival and enhancer (HB9) or promoter (GFAP)-driven lineage differentiation of transplanted cells. We hypothesize that accurate cell injections, extended migration distances, and the relative ratio of cell survival and downstream cell differentiation will correlate with the measured behavioral scores, electrophysiologic readouts, and total skeletal muscle mass segmentation measurements. In addition, we will test the feasibility of our novel artificial MRI reporter, lysine-rich protein (LRP) or its improved derivatives, to interrogate the fate of cells in a similar fashion as using the luciferase gene. PUBLIC HEALTH RELEVANCE We will use magnetic resonance imaging and bioluminescence imaging as non-invasive imaging techniques to monitor the distribution, survival, and fate of transplanted stem or progenitor cells in pre-clinical animal models of motor neuron disease that resemble Lou Gehrig's disease, a devastating disease for which there is no cure. The ultimate goal is to develop ways of reporting on successful cell transplantation without removing any tissue, and to provide neurologists with a means to evaluate stem cell treatment in their patients.

A major parameter limiting immune responses to vaccination is the number of activated antigen presenting cells (APCs) that capture antigen from the vaccine site and migrate to draining lymph nodes (LNs), the site where T and B cell priming occurs. Currently, a quantitative non-invasive technique for monitoring in vivo antigen capture and dehvery is laclting. The use of cellular magnetic resonance imaging (MRI) is a promising approach for this purpose;however, cellular imaging currently requires ex vivo pre-labeling of cells with contrast agents followed by reintroduction of cells into the subject being monitored. Using mouse models, we have developed an in vivo labehng method which relies upon the capture of vaccine antigen-associated superparamagnetic iron oxide (SPIO) by endogenous antigen presenting cells, in situ, in order to quantify AFC delivery to LNs. In this system, MRI is capable of monitoring the trafficking of magnetically labeled APCs in vivo that are responsible for inducing tumor-specific immune responses. Analysis of lymph node MR images using dedicated software enables signal quantification through the generation of pixel intensity histograms. Excellent correlation is observed between in vivo and ex vivo quantification of vaccine antigen-loaded APCs, with resolution sufficient to detect increased APC trafficking elicited by an adjuvant. Furthermore, APCs that capture SPIO (and antigen) and traffic to LN can subsequently be magnetically recovered ex vivo, allowing for detailed cellular and molecular studies of the upstream parameters that influence the afferent arm of vaccine-induced immunity. Using murine vaccine models targeting lung cancer, we now propose to examine the correlation between the extent of APC trafficking as measured by MRI and the quantity and quality of the downstream immune response. This information will be used to evaluate candidate vaccine adjuvants for their ability to augment this critical step In generating systemic immunity. We will also extend this technology to additional vaccine platforms setting the stage for clinical application.

Metal ions play a crucial role in myriad biological processes, and the ability to monitor real-time changes in metal ion levels is essential for understanding a variety of physiological events. Although imaging of dynamic changes in metal ion levels is applicable in vitro using optical-based responsive dyes, which are limited by low tissue penetration, noninvasive detection of free metal ions in vivo in deep tissues remains a formidable challenge. MRI-based sensors allow the longitudinal study of the same subject with unlimited tissue penetration, which may be useful for studying dynamic biological processes, such as the occurrence and progression of diseases and the efficacy of suggested therapeutics. Moreover, and importantly, MRI sensors have the potential for clinical translation. This proposal aims to design, synthesize, characterize, and optimize fluorinated chelates for Zn2+ imaging using a recently developed approach called ion Chemical Exchange Saturation Transfer (iCEST)-MRI. We have recently shown that using a fluorinated derivative of a calcium chelate, 5,52-difluoro BAPTA (5F-BAPTA), allows the detection of low, biologically relevant concentrations of calcium using iCEST. As a first step, a series of potential iCEST sensors for Zn2+ imaging will be synthesized and studied to characterize their ability to monitor labile zinc ions. Properties such as the dissociation constant (Kd), the frequency offset between bound and free chelate in the 19F-NMR spectrum ( ¿), the exchange rate (kex), the specificity for Zn2+ (compared to competitive ions), and contrast enhancement, which are all critical for the design of iCEST agents, will be examined. Then, the limitations of the preferred probes, such as the detectability levels of the probe and the dynamic range of detectable Zn2+ concentrations, will be examined. As an example of a potential application, we will capitalize on the ample experience of our research group in pancreatic islet cells as a therapeutic strategy for diabetes. Since insulin secretion from pancreatic beta cells is accompanied by a high concentration of zinc that is released into the extracellular space, monitoring Zn2+ is considered a biomarker for pancreatic beta cell function. We will monitor the Zn2+ release from therapeutic pancreatic islet cells upon the addition of glucose and correlate these levels (detected by the iCEST methodology) with insulin secretion, i.e., their functionality and therapeutic capabilities. Upon completion of this study, we anticipate establishing a new approach to imaging labile Zn2+ in biological systems using MRI, which will be applicable to a broad spectrum of biomedical applications and will launch a novel strategy for imaging biologically relevant metal ions.

? DESCRIPTION (provided by applicant): For patients with Type I diabetes mellitus, islet transplantation provides a moment-to-moment fine regulation of insulin that is unachievable by exogenous insulin injection. Islet cell death, caused either by transplant rejection, the presence of toxic immunosuppressive drugs, and/or the lack of blood and nutrient supply remains an important obstacle for successful therapy. For some time, advances have been made by encapsulating islets with alginate to achieve immunoprotection, but the overall success rate has been limited, with poor long-term survival of islets once transplanted into patients. From biopsies it is known that the currently used alginate capsule compositions are far from optimal, as they can elicit a foreign body host immune response culminating in fibrotic overgrowth of capsules and subsequent islet cell death. Unfortunately, there is currently no means to probe the mechanical stability and biocompatibility of engrafted microcapsules non-invasively over time, delaying further development and improvements of encapsulated islet cell therapy. Our goal is to develop a dual-mode magnetic resonance imaging (MRI) approach that can report on the mechanical stability of implanted capsules over time while simultaneously interrogating the absence or presence of a major host immune response. To this end, we will employ fluorine (19F) and magnetization transfer (MT) MRI, respectively, both of which are clinically available. Mixed Alginate Gradient (MAG) fluorocapsules will be developed as a new capsule formulation without the need for using potentially toxic polycations to achieve selectivity of capsule permeability. We hypothesize that these MAG fluorocapsules have improved biocompatibility profiles over conventional alginate encapsulants. We will first develop MAG fluorocapsules with different mechanical strengths, and test their stability, perm-selectivity, and islet- encapsulated functionality in vitro. We will then transplant empty capsules s.c. and i.p. in non-diabetic, immunocompetent Balb/c mice which will be followed for 180 days. The outcome of the MRI studies and immunohistopathology will be used to select the most promising formulation to encapsulate mouse (allogeneic) and porcine and human (xenogeneic) islets, which will be transplanted i.p. and s.c. in immunocompetent NOD Shi/Ltj and streptozotocin (STZ)-induced immunodeficient NOD scid/scid mice. 19F MRI (mechanical stability) and MT MRI (host immune response) signals will be collected over 180 days and compared to immunohistological and blood (c-peptide, glucose) parameters. The relative islet cell survival will be quantified with bioluminescent imaging (BLI) and correlated to the fluorine and MTR signals. By following a step-wise approach of allografting and xenografting islets from different sources, with increasing demand on immunoprotection, biocompatibility, and preservation of mechanical stability, we hope to demonstrate the usefulness of dual-mode MRI in developing novel encapsulating materials with potential for clinical translation.

Hydrogel scaffolds are increasingly being used as tissue-mimicking materials and as vehicles to improve transplanted stem cell retention and survival. We have recently developed a new chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method that is able to probe the in vivo stability and gelatin decomposition of implanted composite hyaluronic acid (HA) hydrogels in a ?label-free? fashion. Compared to naked cells, we found that transplanted neural stem cells showed improved survival when hydrogel scaffolding was applied. A major question that remains is the optimal mechanical properties of the hydrogel, and how this relates to cell survival. At the one hand, for initial structural support, the gels should not decompose too fast, but at the other hand they should at some point decompose to allow transplanted cells to grow out and integrate with the surrounding host tissue. Our aim is to synthesize a range of composite near- infrared (NIR)-HA hydrogels with different compositions and stabilities, to image their stability properties in vivo, and to correlate the CEST/NIR optical imaging findings with cell survival as assessed using bioluminescent imaging (BLI) as conventional readout. Using supercharged green fluorescent protein (sGFP) as a new CEST MRI bimodal reporter gene, we will also investigate whether or not CEST MRI is able to probe in vivo cell survival simultaneously. We have chosen to apply this approach to transplantation of glial-restricted precursor cells (GRPs) in a transgenic amyotropic lateral sclerosis (ALS) mouse model, as we have found that transplanted naked cells without hydrogel scaffolding survive poorly in the hostile ALS tissue environment.

SUMMARY for TR&D 2 TR&D 2 of the BTRC will develop novel synthetic, non-metallic reporter genes that can be detected with chemical exchange saturation transfer (CEST) MRI for precise visualization and pinpointing of biological processes in living organisms. Using a lysine-rich protein (LRP) as such a prototype gene, we have previously demonstrated that 1) We can image rapidly dividing tumor cells without the limitation of a label dilution effect that currently exists with conventional MR contrast agents; 2) We can image promoter-driven specific gene expression; and 3) we can image oncolytic virotherapy. In this TR&D, we aim to dramatically improve the CEST contrast and biocompatibility of LRP for further dissemination to the scientific community, and to create a pathway towards eventual clinical translation. Using advanced, rational design-based molecular-genetic engineering approaches, we will first develop a so-called ?enhanced? LRP, or eLRP (Aim 1a). Enhancement is defined by transcription and translation efficiency, protein refolding, optimal proton exchange rate, and strongest CEST contrast. For the latter, a custom-designed high-throughput screening methodology will be used to determine optimal peptide sequence configurations. Next, we will ?humanize? eLRP to create heLRP, using an array of immunological assays (Aim 1b). We will use established algorithms to identify epitopes that induce a T cell and/or humoral response in reverse. Re-engineered LRPs will undergo reiterated screening processes until all immunogenicity has been eliminated without compromising CEST contrast. Alternatively, we will use human protamine-1 (hPRM1) as a starting template to create chimeric LRP/hPRM1 constructs through DNA shuffling. The absence of serum polyclonal antibodies from heLRP-immunized rabbits will be used as a final key criteria for TR&D 2 dissemination. During this immunological screening process, we will simultaneously identify the counterpart of heLRP, i.e., an ?immunogenic? LRP or iLRP. Following in vivo transfection, this iLRP will be used as a new theranostic vector to simultaneously induce an anti-tumor immune response and visualize subsequent tumor cell regression (Aim 2). Finally, we aim to demonstrate how eLRP can be used to provide a unique dynamic insight into biological processes and as defined by cell-cell interactions. We have chosen dendritic cell (DC) immunotherapy as an example. Following a validation study to confirm that constitutively expressed eLRP DCs can be detected in vivo when migrating to lymph nodes following vaccination (Aim 3a), we will investigate when and where DC activation occurs upon presenting antigen to CD4+ cells. We aim to accomplish this using IL-12 promoter-driven specific expression (Aim 3b). Concurrently, we will assess the time course and biodistribution of activated, Ova-specific CD4+ transgenic cells using BLI in an Ova-expressing melanoma mouse model. Our LRP reporters will have many applications in the study of basic cell biology and cell malfunctioning in a wide variety of disease models, as they can be designed de novo and in silico, and hence have unlimited potential for manipulation and fine-tuning as needed for the precise visualization of the biological process in question.

Amyotrophic Lateral Sclerosis (ALS) is a devastating disease with near 100% mortality. An important role has been assigned to (over)activated astrocytes. Given the few available treatment options, stem cell therapy is now actively being pursued as a new treatment paradigm, aimed at either immunomodulation (using mesenchymal stem cells - MSCs), astrocyte replacement (using glial progenitors), or motor neuron restoration, with several clinical trials currently in progress. Our goal is to pursue a two-pronged approach using co- transplantation of both MSCs and glial-restricted precursor cells (GRPs) to achieve simultaneous immunosuppression and glia restoration. Central to our transplantation studies is the development of imaging biomarkers that can non-invasively report not only on the fate of transplanted cells but also on changes in the ALS host environment. To this end, we aim to interrogate 1) the activation state of host cells in vivo using manganese-enhanced magnetic resonance imaging MRI (MEMRI); 2) the in vivo survival of transplanted GRPs using conventional bioluminescent imaging (BLI) as gold standard with 19F MRI cell tracking as an exploratory but clinically applicable surrogate marker; 3) and the disease outcome using time of onset, animal survival, behavioral, and compound muscle action potential (CMAP) measurements. Three transgenic SOD1 and two age-matched wild type cohorts of animals will be enrolled in these studies. Luciferase-transfected and fluorinated hGRPs and unlabeled human MSCs will be transplanted at day 60, i.e., about 30 days before onset of the disease. All animals will be monitored weekly for weight, behavioral scores, and end survival. Randomized groups will undergo BLI, CMAP, and 19F MRI and MEMRI at 1 day, and 1, 2, and 3 months after transplantation up until approximately 150 days. Immunohistological analysis of neuroinflammation, motor neuron degeneration, astrocyte pathology, and grafted cell survival will be used to validate the imaging findings and to compare the potential therapeutic benefit of MSC co-transplantation.

The use of gene-edited stem cells and in particular patient-derived iPSCs for cell replacement therapy is an appealing approach for genetic correction of a disease-associated gene mutation. If such therapies are pursued in future patients, it would be highly desirable to have non-invasive cell tracking techniques available that can report longitudinally on the distribution and survival of transplanted cells, in order to better understand their fate in vivo and to optimize personalized therapies. We have chosen amyotrophic lateral sclerosis (ALS), a devastating disease with near 100% mortality as an example of a target disease, in which loss of motor neurons is a key event leading to muscle paralysis. For familial forms of ALS, mutations in the gene superoxide dismutase 1 (SOD1) are known to be a causative factor for disease development. In this proposal, we aim to apply two novel experimental imaging modalities (MPI and PSMA-targeted 18F-DCFPyL PET) in conjunction with a clinically emerging technique (1H MRI) to answer several basic questions associated with the efficacy and safety of genome-edited cell therapy. These are complementary techniques, where MPI and PSMA- targeted 18F-DCFPyL PET can report on the whole-body distribution of administered cells, whereas MRI can report on real-time homing and immediate retention of cells. This three-pronged approach of imaging the same cell (labeled with SPIO for MPI and MRI, and 18F-DCFPyL for PET) will be applied for tracking of patient- derived native (39b-SOD1+/AV4) and genome-corrected (39b-SOD1+/+) iPSCs, with and without differentiation into motor neurons, in a transgenic SOD1G37R mouse model of ALS. Intra-arterial injection, an emerging cell delivery route, will be used to study the feasibility of real-time image-guided cell injections aimed at obtaining a more global cerebral cell distribution, while intraparenchymal injection in the spinal cord will be applied as a clinically effective delivery technique to deliver cells locally at the site of impaired motor neurons. If successful, this example imaging application of genome-corrected cells in ALS may encourage the use of MPI, MRI, and/or PMSA-based PET imaging to interrogate the fate of cells in other disease scenarios in vivo.

Our overall aim is to develop a precision-based nanotheranostic platform where the imaging signal may serve as an early predictive imaging biomarker for intracellular nanoparticle accumulation and therapeutic response. New anti-cancer agents continue to be developed, but many fail due to the tumor developing (multi-) drug resistance. Cellular membrane proteins acting as a drug efflux pump have been identified, and while some promising agents enter tumor cells, they cannot always be retained long enough to be effective. We aim to exploit the enzyme legumain (an asparaginyl endopeptidase) that is overexpressed in prostate cancer cells for specific cleavage of an olsalazine (Olsa)-conjugated peptide substrate, following which the substrate self- assembles into intracellular nanoparticles. This enzyme-driven self-assembly serves several purposes: 1) intracellular entrapment with minimal drug efflux; 2) prolonged tumor drug exposure; and 3) minimal toxicity to normal organs due to rapid blood clearance of non-assembled single molecules. We have preliminary data demonstrating this concept to be feasible in vivo. Since it does not only serve as an anti-cancer drug through inhibition of DNA methylation, but also as a non-metallic, label-free contrast agent for chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), olsalazine is a unique theranostic agent. The drug can be visualized without modification, allowing direct imaging without pharmacological alterations that may affect self-assembly and/or biodistribution. Following in vitro selection of an optimal Olsa-CBT-800CW-Rn- AAN substrate with maximum tumor cell penetration and retention in legumain-overexpressing DU145 cells (Aim 1), we will test this compound for its in vivo nanotheranostic properties in an orthotopic mouse prostate tumor model (Aim 2) and a transgenic mouse model (TRAMP mouse) where normal prostate cells undergo a malignant transformation over time (Aim 3). If successful, this approach may be extended to other enzyme- targeted CEST MRI-detectable theranostic platforms for imaging tumor aggressiveness, drug accumulation, and predicting therapeutic response.

Precision magnetic hyperthermia by integrating magnetic particle imaging Magnetic activation of magnetic iron oxide nanoparticles (MIONPs) offers considerable potential for numerous biomedical applications. Approved clinical applications include contrast enhancement for magnetic resonance imaging (MRI) and magnetic fluid hyperthermia (MFH) for cancer treatment. MIONPs are T2 negative contrast agents which have been clinically available for MRI since the late 1980s where very low tissue concentrations (<100 ?g Fe/g tissue) are needed for imaging. MFH is a powerful nanotechnology-based treatment that enhances radiation therapy (RT). It comprises local heating of tissue by activating MIONPs with an external alternating magnetic field (AMF), enabling treatment anywhere in the body. Human clinical trials demonstrated benefits of MFH for prostate cancer; and, overall survival benefits with RT in recurrent glioblastoma (GBM) resulted in European approval in 2010. However, current MFH effectiveness is limited by the inability to visualize MIONP distribution during MFH, resulting in poor AMF control of MIONP heating, reduced therapeutic efficacy, and unwanted off-target toxicity. An integrated MIONP imaging-MFH technology that provides spatial control of the MFH treatment volume will substantially advance the clinical use of theranostic MIONPs. Magnetic particle imaging (MPI) is an emerging imaging technology that directly quantitates MIONP concentration in tissue with similar or greater sensitivity as MRI. The main magnet in an MPI scanner produces a strong magnetic field gradient containing a region where the magnetic field is approximately zero, i.e. the Field Free Region (FFR). MIONPs in the FFR are magnetically unsaturated and can produce a signal in a receiver coil, while MIONPs elsewhere are magnetically saturated and produce no signal. Images are produced by rastering the FFR across the sample. The FFR used for imaging can be used to localize MFH. By applying a magnetic field gradient and AMF, only MIONPs inside the FFR will heat while MIONPs outside the FFR are saturated and do not heat. MPI and MFH are compatible enabling mm-precision spatial control of MFH. Our objective is to develop an integrated MPI/MFH workflow that incorporates imaging-guided treatment planning with optimal theranostic MIONPs for preclinical biomedical research with small animal (mouse and rat) models. We aim to achieve our objectives by purchasing a HYPER AMF system that will be used with our recently acquired Momentum MPI scanner (funded by a S10 shared instrumentation grant). Our specific aims are: (Aim 1) Identify MIONPs having ideal physical and magnetic properties for MPI/MFH; (Aim 2) Develop MPI-guided MFH treatment using computational modeling and amplitude modulation; (Aim 3) Demonstrate increased therapeutic efficacy of theranostic MPI/MFH in vivo. While the primary objective of the proposed effort is technology development, successful completion of the aims will provide biomedical researchers the ability to realize theranostic applications with magnetic nanoparticles.