Biology subjects

Ronald, J. A.

Publications and source records attributed to Ronald, J. A..

13 recordsLinked to original sources

bioRxiv2025

Imaging CRISPR-Edited CAR-T Cell Therapies with Optical and Positron Emission Tomography Reporters

RationaleChimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. MethodsThe luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. ResultsT cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70-80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naive T cells. In vivo, AkaLuc BLI and 18F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. ConclusiontRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.

immunology↗

bioRxiv2024

Probing the effects of polysaccharide hydrogel composition on the viability and pro-angiogenic function of human adipose-derived stromal cells

Cell therapies harnessing the pro-vascular regenerative capacities of mesenchymal stromal cell (MSC) populations, including human adipose-derived stromal cells (hASCs), have generated considerable interest as an emerging treatment strategy for peripheral arterial disease (PAD) and its progression to critical limb ischemia (CLI). There is evidence to support that polysaccharide hydrogels can enhance therapeutic efficacy when applied as minimally-invasive delivery systems to support MSC survival and retention within ischemic tissues. However, there has been limited research to date on the effects of hydrogel composition on the phenotype and function of encapsulated cell populations. Recognizing this knowledge gap, this study compared the pro-angiogenic function of hASCs encapsulated in distinct but similarly-modified natural polysaccharide hydrogels composed of methacrylated glycol chitosan (MGC) and methacrylated hyaluronic acid (MHA). Initial in vitro studies confirmed high viability (>85%) of the hASCs following encapsulation and culture in the MGC and MHA hydrogels over 14 days, with a decrease in the cell density observed over time. Moreover, higher levels of a variety of secreted pro-angiogenic and immunomodulatory factors were detected in conditioned media samples collected from the hASCs encapsulated in the MGC-based hydrogels compared to the MHA hydrogels. Subsequent testing focused on comparing hASC delivery within the MGC and MHA hydrogels to saline controls in a femoral artery ligation-induced CLI (FAL-CLI) model in athymic nu/nu mice over 28 days. For the in vivo studies, the hASCs were engineered to express tdTomato and firefly luciferase to quantitatively compare the efficacy of the two platforms in supporting the localized retention of viable ASCs through longitudinal cell tracking with bioluminescence imaging (BLI). Interestingly, hASC retention was significantly enhanced when the cells were delivered in the MHA hydrogels as compared to the MGC hydrogels or saline. However, laser Doppler perfusion imaging (LDPI) indicated that the restoration of hindlimb perfusion was similar between the treatment groups and controls. These findings were corroborated by endpoint immunofluorescence (IF) staining showing similar levels of CD31+ cells in the ligated limbs at 28 days in all groups. Overall, this study demonstrates that enhanced MSC retention may be insufficient to augment vascular regeneration, emphasizing the complexity of designing biomaterials platforms for MSC delivery for therapeutic angiogenesis. In addition, the data points to a potential challenge in approaches that seek to harness the paracrine functionality of MSCs, as strategies that increase the secretion of immunomodulatory factors that can aid in regeneration may also lead to more rapid MSC clearance in vivo.

bioengineering↗

bioRxiv2023

A synthetic Notch (synNotch) system linking intratumoral immune-cancer cell communication to a synthetic blood biomarker assay

IntroductionCellular immunotherapy has greatly improved cancer treatment in recent years. For instance, chimeric antigen receptor (CAR) T-cell therapy has been proven highly effective in treating hematological malignancies, and many CAR cell designs are being explored for solid tumors. However, many questions remain why responses differ across patients and some tumor types are resistant. Improved and relatively inexpensive ways to monitor these cells could provide some answers. Clinically, blood tests are regularly used to monitor these therapies, but blood signals often do not reflect the activity of immune cells within the tumor(s). Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test. Specifically, we engineered immune cells with a CD19-targeted synNotch receptor and demonstrate that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP) at levels that are readily detected in the blood. Methods and ResultsJurkat T cells were engineered via sequential lentiviral transduction of two components: an anti-CD19 synNotch receptor and asynNotchresponse element encoding SEAP. Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media. Nod-scid-gamma (NSG) mice were subcutaneously injected with either CD19+ or CD19- Nalm6 cells. Intratumoral injection of engineered T cells (1x107) resulted significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n=7), but not CD19- tumors (n=5). DiscussionOur synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test. Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies during their initial development and clinical translation, ultimately improving our understanding of design considerations and patient-specific responses.

synthetic biology↗

bioRxiv2023

Delivery of human adipose-derived stromal cells within mechanically resilient hydrogels induces adverse outcomes in a femoral artery ligation model in athymic nu/nu mice

The delivery of human adipose-derived stromal cells (hASCs) to ischemic tissues represents a promising strategy to promote vascular regeneration for patients with critical limb ischemia (CLI). Building on previous work, this study focused on the in vivo characterization of a hydrogel cell delivery platform for hASCs composed of peptide-functionalized methacrylated glycol chitosan (MGC-RGD) and a terminally acrylated triblock copolymer of poly(ethylene glycol) and poly(trimethylene carbonate) (PEG(PTMC-A)2) in athymic nu/nu mice with femoral artery ligation-induced critical limb ischemia (FAL-CLI). This immunodeficient mouse strain was selected to enable human cell transplantation in a model with conserved monocyte/macrophage function, recognizing that macrophages are key regulators of the biomaterial implant response, as well as vascular repair and regeneration. The hASCs were engineered to co-express tdTomato and firefly luciferase to enable longitudinal cell tracking using bioluminescence imaging (BLI). Interestingly, the hASCs were better retained following delivery in saline compared to hydrogel delivery. However, laser Doppler perfusion imaging (LDPI) analysis indicated that the restoration of hindlimb perfusion was similar between the two cell treatment groups. Critically, delivery of the hASCs within the hydrogels was associated with adverse outcomes only observed within this treatment group, including severe swelling, discoloration, and necrosis, which necessitated early euthanasia of some mice. CD45 staining supported that the combination of the cells and the hydrogels induced an inflammatory host response. These findings contrast with previous positive results when the platform was tested for hASC delivery in more severely immunocompromised NOD/SCID mice with FAL-CLI, as well as allogeneic rat ASC delivery in a healthy immunocompetent rat model. Overall, this study emphasizes the potential importance of testing cell delivery platforms in pre-clinical disease models that have retained host immune cell function, especially for immunomodulatory cell populations such as ASCs.

bioengineering↗

bioRxiv2022

Sensitive Spatiotemporal Tracking of Spontaneous Metastasis in Deep Tissues via a Genetically-Encoded Magnetic Resonance Imaging Reporter

Metastasis remains a poorly understood aspect of cancer biology and the leading cause of cancer-related death, yet most preclinical cancer studies do not examine metastasis, focusing solely on the primary tumor. One major factor contributing to this paradox is a gap in available tools for accurate spatiotemporal measurements of metastatic spread in vivo. Our objective was to develop an imaging reporter system that offers sensitive three-dimensional detection of cancer cells at high resolutions in live mice. We utilized organic anion-transporting polypeptide lb3 (oatp1b3) as a magnetic resonance imaging (MRI) reporter gene to this end, and systematically optimized its framework for in vivo tracking of viable cancer cells in a spontaneous metastasis model. We were able to image metastasis on oatp1b3-MRI at the single lymph node level and continued to track its progression over time as cancer cells spread to multiple lymph nodes and different organ systems in single animals. While initial single lesions were successfully imaged in parallel via bioluminescence, later metastases were obscured by light scatter from the initial node. Importantly, we demonstrate and validate that 100-m isotropic resolution MR images could detect micrometastases in lung tissue estimated to contain fewer than 103 cancer cells. In summary, oatp1b3-MRI enables precise determination of lesion size and location over time and offers a path towards deep-tissue tracking of any oatp1b3-engineered cell type with combined high resolution, high sensitivity, 3D spatial information, and surrounding anatomical context.

bioengineering↗

bioRxiv2021

Gadolinium-free Magnetic Resonance Imaging of the Liver using an Oatp1-targeted Manganese(III) Porphyrin

Controversy surrounding gadolinium-based contrast agents (GBCAs) have rendered their continued utility highly contentious, but the liver-specific GBCA Gd(III) ethoxybenzyl-diethylene triamine pentaacetic acid (Gd(III)-EOB-DTPA) remains in use because it provides unique diagnostic information that could not be obtained by any other means. To address the need for an alternative liver-specific MRI contrast agent, we synthesized Mn(III) 20-(4-ethoxyphenyl) porphyrin-5,10,15-tricarboxylate (Mn(III)TriCP-PhOEt), which exhibited significantly higher r1 relaxivity than Gd(III)-EOB-DTPA, and targeted organic anion-transporting polypeptide 1 (Oatp1) channels as a biomarker of hepatocyte viability. Mn(III)TriCP-PhoEt increased the r1 relaxation rate of cells expressing rodent Oatp1a1 and human Oatp1b3, relative to control cells not expressing these liver channels. In mice, Mn(III)TriCP-PhoEt resulted in significant and specific increases in liver signal intensity on T1-weighted images, and significant decreases in liver T1 time relative to precontrast measurements. Our findings suggest that Mn(III)TriCP-PhOEt operates as a specific and sensitive MR contrast agent for in vivo liver imaging.

biophysics↗

bioRxiv2021

A method for the efficient iron-labeling of patient-derived xenograft cells and cellular imaging validation

There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumour behavior, and metastatic properties observed in the original tumour. These models are more predictive of clinical outcomes and are superior to cell lines for preclinical drug evaluation and therapeutic strategies. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metastases and validate this method with imaging during tumour development. Luciferase expressing human breast cancer PDX cells (F2-7) were successfully labeled after incubation with micron-sized iron oxide particles (MPIO; 25 g Fe/mL). NOD/SCID/ILIIrg-/- (n=5) mice received injections of 1x106 iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. In vivo BLI revealed that signal increased over time with tumour development. MPI revealed decreasing signal in the tumours and increasing signal in the liver region over time. Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumour. To accomplish this, we also demonstrate the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.

biophysics↗

bioRxiv2020

Molecular imaging reveals a high degree of cross-seeding of spontaneous metastases in a novel mouse model of synchronous bilateral breast cancer

Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. However, the lack of a preclinical model has led to a dearth in knowledge regarding the patterns of SBBC metastasis. Here we established an SBBC model and used molecular imaging to visualize the development of spontaneous lung metastases arising from each primary tumor. We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI), and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10) and BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27% to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%. These results reveal a high degree of metastatic cross-seeding which may contribute to faster metastatic growth and intratumoral heterogeneity. We posit our work will help understand treatment resistance and optimal planning of treatment for SBBC patients.

cancer biology↗

bioRxiv2020

A survivin-driven tumour-activatable minicircle system for prostate cancer theranostics

Gene vectors driven by tumour-specific promoters to express reporter genes and therapeutic genes are an emerging approach for improved cancer diagnosis and treatment. Minicircles (MCs) are shortened plasmids stripped of prokaryotic sequences and have potency and safety characteristics that are beneficial for clinical translation. We previously developed survivin-driven, tumour-activatable MCs for cancer detection via a secreted blood reporter assay. Here we present a novel theranostic system for prostate cancer featuring a pair of survivin-driven MCs, combining selective detection of aggressive tumours via a urinary reporter test and subsequent tumour treatment with gene-directed enzyme prodrug therapy. MethodsWe engineered both diagnostic and therapeutic survivin-driven MCs expressing Gaussia luciferase, a secreted reporter that is detectable in the urine, and cytosine deaminase:uracil phosphoribosyltransferase fusion, respectively. Diagnostic MCs were evaluated in mice carrying orthotopic prostate tumours with varying survivin levels, measuring reporter activity in serial urine samples. Therapeutic MCs were evaluated in mice receiving prodrug using bioluminescence imaging to assess cancer cell viability over time. ResultsDiagnostic MCs revealed mice with aggressive prostate tumours exhibited significantly higher urine reporter activity than mice with non-aggressive tumours and tumour-free mice. Combined with 5-fluorocytosine prodrug treatment, therapeutic MCs resulted in reduced bioluminescence signal in mice with aggressive prostate tumours compared to control mice. ConclusionSequential use of these MCs may be used to first identify patients carrying aggressive prostate cancer by a urinary reporter test, followed by stringent treatment in stratified individuals identified to have high-risk lesions. This work serves to highlight tumour-activatable MCs as a viable platform for development of gene-based tumour-activatable theranostics.

synthetic biology↗

bioRxiv2020

Visualizing tumour self-homing with magnetic particle imaging

Due to their innate tumour homing capabilities, in recent years, CTCs have been engineered to express therapeutic genes for targeted treatment of primary and metastatic lesions. Additionally, previous studies have incorporated optical or PET imaging reporter genes to enable noninvasive monitoring of therapeutic CTCs in preclinical tumour models. Here, we demonstrate for the first time, the ability of magnetic particle imaging (MPI) to sensitively detect systemically administered iron-labeled CTCs and to visualize tumour self-homing in a murine model of human breast cancer.

biophysics↗

bioRxiv2020

A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

Imaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 {micro}l subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.

bioengineering↗

bioRxiv2019

Engineering self-homing circulating tumour cells as novel cancer theranostics

Purpose: New ways to target and treat metastatic disease are urgently needed. Tumor \"self-homing\" describes the recruitment of circulating tumor cells (CTCs) back to a previously excised primary tumor location, contributing to tumor recurrence, as well as their migration to established metastatic lesions. Recently, self-homing CTCs have been exploited as delivery vehicles for anti-cancer therapeutics in preclinical primary tumor models. However, the ability of CTCs to self-home and treat metastatic disease is largely unknown. Methods: Here, we employ molecular imaging to explore whether systemically-administered CTCs home to metastatic lesions and if CTCs armed with both a reporter gene and a cytotoxic prodrug gene therapy can be used to visualize and treat metastatic disease. Results: Bioluminescence imaging (BLI) performed over time revealed a remarkable ability of CTCs to home to primary and metastatic tumors throughout the body. Mice that received therapeutic CTCs had less BLI signal as well as less primary tumour burden than control mice. Preliminary data also showed self-homing therapeutic CTCs may be effective at treating disseminated breast cancer metastases. Conclusion: Using dual-luciferase BLI, this study demonstrates the noteworthy ability of experimental CTCs to home to disseminated breast cancer lesions. Moreover, by incorporating a prodrug gene therapy system into our self-homing CTCs, we show exciting progress towards effective and targeted delivery of gene-based therapeutics to treat both primary and metastatic lesions.

cancer biology↗

bioRxiv2019

A human photoacoustic imaging reporter gene using the clinical dye indocyanine green.

Photoacoustic imaging (PAI) combines optical contrast with the resolution and depth-detection of ultrasound and is increasingly being utilized for medical imaging in patients. PAI reporter genes would allow for monitoring of cell and gene therapies, but current reporters have immunogenicity and/or toxicity concerns that may limit clinical translation. Here we report a PAI reporter system employing the ability of human organic anion transporting polypeptide 1b3 (Oatp1b3) to take up the clinical dye indocyanine green (ICG) into cells. Following ICG administration, cells synthetically expressing Oatp1b3 exhibited significantly increased PAI signals compared to control cells both in vitro and in mice. Several benefits of this technology are the human derivation of Oatp1b3, and the high extinction coefficient, low quantum yield and pre-existing clinical approval of ICG. We posit that the Oatp1b3-ICG reporter system could be useful for in vivo gene and cell tracking in preclinical and clinical applications.

bioengineering↗