Katherine Ferrara, Postdoctoral Faculty Sponsor
Protocol for in vitro sonoporation validation using non-targeted microbubbles for human studies of ultrasound-mediated gene delivery.
2023; 4 (4): 102723
Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use. For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1.
View details for DOI 10.1016/j.xpro.2023.102723
View details for PubMedID 37976155
Sonogenetic control of multiplexed genome regulation and base editing.
2023; 14 (1): 6575
Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.
View details for DOI 10.1038/s41467-023-42249-8
View details for PubMedID 37852951
View details for PubMedCentralID PMC10584809
PET imaging of focused-ultrasound enhanced delivery of AAVs into the murine brain.
2023; 13 (15): 5151-5169
Rationale: Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. Methods: In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for in vivo PET quantification, whereas results for ex vivo assays are reported as percent injected dose per gram (%ID/g). Results: In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed in vivo by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[18F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Conclusion: Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies.
View details for DOI 10.7150/thno.85549
View details for PubMedID 37908737
View details for PubMedCentralID PMC10614693
Multiomic analysis for optimization of combined focal and immunotherapy protocols in murine pancreatic cancer.
2022; 12 (18): 7884-7902
Background: Although combination immunotherapies incorporating local and systemic components have shown promising results in treating solid tumors, varied tumor microenvironments (TMEs) can impact immunotherapeutic efficacy. Method: We designed and evaluated treatment strategies for breast and pancreatic cancer combining magnetic resonance-guided focused ultrasound (MRgFUS) ablation and antibody therapies. With a combination of single-cell sequencing, spectral flow cytometry, and histological analyses, we profiled an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a dense epithelial neu deletion (NDL) HER2+ mammary adenocarcinoma model with a greater fraction of lymphocytes, natural killer cells and activated dendritic cells. We then performed gene ontology analysis, spectral and digital cytometry to assess the immune response to combination immunotherapies and correlation with survival studies. Result: Based on gene ontology analysis, adding ablation to immunotherapy enriched immune cell migration pathways in the pancreatic cancer model and extensively enriched wound healing pathways in the breast cancer model. With CIBERSORTx digital cytometry, aCD40 + aPD-1 immunotherapy combinations enhanced dendritic cell activation in both models. In the MT4 TME, adding the combination of aCD40 antibody and checkpoint inhibitors (aPD-1 and aCTLA-4) with ablation was synergistic, increasing activated natural killer cells and T cells in distant tumors. Furthermore, ablation with immunotherapy upregulated critical Ly6c myeloid remodeling phenotypes that enhance T-cell effector function and increased granzyme and protease encoding genes by as much as 100-fold. Ablation combined with immunotherapy then extended survival in the MT4 model to a greater extent than immunotherapy alone. Conclusion: In summary, TME profiling informed a successful multicomponent treatment protocol incorporating ablation and facilitated differentiation of TMEs in which ablation is most effective.
View details for DOI 10.7150/thno.73218
View details for PubMedID 36451859
View details for PubMedCentralID PMC9706583
Optimization of microbubble-based DNA vaccination with low-frequency ultrasound for enhanced cancer immunotherapy.
2021; 4 (9)
Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, we optimized a strategy employing low ultrasound transmission frequency-induced bubble oscillation to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles. The diameter of the small microbubble population was 1.27 ± 0.89 μm and that of larger population was 4.23 ± 2.27 μm. Upon in vitro insonation with the larger microbubble population, 29.7% of cancer cells were transfected with DNA plasmids, higher than that with smaller microbubbles (18.9%, P <0.05) or positive control treatments with a commercial transfection reagent (12%, P < 0.01). After 48 h, gene expression increased more than two-fold in tumors treated with large, as compared with small, microbubbles. Furthermore, the immune response, including tumor infiltration of CD8+ T cells and F4/80+ macrophages, was enhanced. We believe that this safe and efficacious method can improve preclinical procedures and outcomes for DNA vaccines in cancer immunotherapy in the future.
View details for DOI 10.1002/adtp.202100033
View details for PubMedID 34632048
View details for PubMedCentralID PMC8494128
Synergies between therapeutic ultrasound, gene therapy and immunotherapy in cancer treatment.
Advanced drug delivery reviews
Due to the ease of use and excellent safety profile, ultrasound is a promising technique for both diagnosis and site-specific therapy. Ultrasound-based techniques have been developed to enhance the pharmacokinetics and efficacy of therapeutic agents in cancer treatment. In particular, transfection with exogenous nucleic acids has the potential to stimulate an immune response in the tumor microenvironment. Ultrasound-mediated gene transfection is a growing field, and recent work has incorporated this technique into cancer immunotherapy. Compared with other gene transfection methods, ultrasound-mediated gene transfection has a unique opportunity to augment the intracellular uptake of nucleic acids while safely and stably modulating the expression of immunostimulatory cytokines. The development and commercialization of therapeutic ultrasound systems further enhance the potential translation. In this Review, we introduce the underlying mechanisms and ongoing preclinical studies of ultrasound-based techniques in gene transfection for cancer immunotherapy. Furthermore, we expand on aspects of therapeutic ultrasound that impact gene therapy and immunotherapy, including tumor debulking, enhancing cytokines and chemokines and altering nanoparticle pharmacokinetics as these effects of ultrasound cannot be fully dissected from targeted gene therapy. We finally explore the outlook for this rapidly developing field.
View details for DOI 10.1016/j.addr.2021.113906
View details for PubMedID 34333075
- Optimization of Microbubble-Based DNA Vaccination with Low-Frequency Ultrasound for Enhanced Cancer Immunotherapy ADVANCED THERAPEUTICS 2021
Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer.
2021; 11 (1): 927
High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65°C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1beta and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.
View details for DOI 10.1038/s41598-020-80135-1
View details for PubMedID 33441763
Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites.
Proceedings of the National Academy of Sciences of the United States of America
Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.
View details for DOI 10.1073/pnas.1914906117
View details for PubMedID 32430322