Bacterial therapies at the interface of synthetic biology and nanomedicine.

Bacteria are emerging as living drugs to treat a broad range of disease indications. However, the inherent advantages of these replicating and immunostimulatory therapies also carry the potential for toxicity. Advances in synthetic biology and the integration of nanomedicine can address this challenge through the engineering of controllable systems that regulate spatial and temporal activation for improved safety and efficacy. Here, we review recent progress in nanobiotechnology-driven engineering of bacteria-based therapies, highlighting limitations and opportunities that will facilitate clinical translation.

"Tumor-selective treatment of metastatic pancreatic cancer with an engineered, probiotic living drug".

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges for effective treatment, with systemic chemotherapy often proving inadequate due to poor drug delivery and the tumor's immunosuppressive microenvironment. Engineered bacteria present a novel approach to target PDAC, leveraging their ability to colonize tumors and deliver therapeutic payloads. Here, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce the pore-forming Theta toxin (Nis-Theta) and evaluated its efficacy in a preclinical model of PDAC. Probiotic administration resulted in selective colonization of tumor tissue, leading to improved overall survival compared to standard chemotherapy. Moreover, this strain exhibited cytotoxic effects on both primary and distant tumor lesions while sparing normal tissues. Importantly, treatment also modulated the tumor microenvironment by increasing anti-tumor immune cell populations and reducing immunosuppressive markers. These findings demonstrate the potential of engineered probiotic bacteria as a safe and effective therapeutic approach for PDAC, offering promise for improved patient outcomes.

Design of combination therapy for engineered bacterial therapeutics in non-small cell lung cancer.

Synthetic biology enables the engineering of bacteria to safely deliver potent payloads to tumors for effective anti-cancer therapies. However, a central challenge for translation is determining ideal bacterial therapy candidates for specific cancers and integrating them with other drug treatment strategies to maximize efficacy. To address this, we designed a screening and evaluation pipeline for characterization of bacterial therapies in lung cancer models. We screened 10 engineered bacterial toxins across 6 non-small cell lung cancer patient-derived cell lines and identified theta toxin as a promising therapeutic candidate. Using a bacteria-spheroid co-culture system (BSCC), analysis of differentially expressed transcripts and gene set enrichment revealed significant changes in at least 10 signaling pathways with bacteria-producing theta toxin. We assessed combinatorial treatment of small molecule pharmaceutical inhibitors targeting 5 signaling molecules and of 2 chemotherapy drugs along with bacterially-produced theta toxin and showed improved dose-dependent response. This combination strategy was further tested and confirmed, with AKT signaling as an example, in a mouse model of lung cancer. In summary, we developed a pipeline to rapidly characterize bacterial therapies and integrate them with current targeted therapies for lung cancer.

A rapid screening platform to coculture bacteria within tumor spheroids.

The prevalence of tumor-colonizing bacteria along with advances in synthetic biology are leading to a new generation of living microbial cancer therapies. Because many bacterial systems can be engineered to recombinantly produce therapeutics within tumors, simple and high-throughput experimental platforms are needed to screen the large collections of bacteria candidates and characterize their interactions with cancer cells. Here, we describe a protocol to selectively grow bacteria within the core of tumor spheroids, allowing for their continuous and parallel profiling in physiologically relevant conditions. Specifically, tumor spheroids are incubated with bacteria in a 96-well low-adhesion plate followed by a series of washing steps and an antibiotic selection protocol to confine bacterial growth within the hypoxic and necrotic core of tumor spheroids. This bacteria spheroid coculture (BSCC) system is stable for over 2 weeks, does not require specialized equipment and is compatible with time-lapse microscopy, commercial staining assays and histology that uniquely enable analysis of growth kinetics, viability and spatial distribution of both cellular populations, respectively. We show that the procedure is applicable to multiple tumor cell types and bacterial species by varying protocol parameters and is validated by using animal models. The BSCC platform will allow the study of bacteria-tumor interactions in a continuous manner and facilitate the rapid development of engineered microbial therapies.

A programmable encapsulation system improves delivery of therapeutic bacteria in mice.

Living bacteria therapies have been proposed as an alternative approach to treating a broad array of cancers. In this study, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides that enhances systemic delivery. Based on a small RNA screen of capsular biosynthesis pathways, we constructed inducible synthetic gene circuits that regulate bacterial encapsulation in Escherichia coli Nissle 1917. These bacteria are capable of temporarily evading immune attack, whereas subsequent loss of encapsulation results in effective clearance in vivo. This dynamic delivery strategy enabled a ten-fold increase in maximum tolerated dose of bacteria and improved anti-tumor efficacy in murine models of cancer. Furthermore, in situ encapsulation increased the fraction of microbial translocation among mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system promises to enhance the therapeutic utility of living engineered bacteria for cancer.

Enhancing the tropism of bacteria via genetically programmed biosensors.

Engineered bacteria for therapeutic applications would benefit from control mechanisms that confine the growth of the bacteria within specific tissues or regions in the body. Here we show that the tropism of engineered bacteria can be enhanced by coupling bacterial growth with genetic circuits that sense oxygen, pH or lactate through the control of the expression of essential genes. Bacteria that were engineered with pH or oxygen sensors showed preferential growth in physiologically relevant acidic or oxygen conditions, and reduced growth outside the permissive environments when orally delivered to mice. In syngeneic mice bearing subcutaneous tumours, bacteria engineered with both hypoxia and lactate biosensors coupled through an AND gate showed increased tumour specificity. The multiplexing of genetic circuits may be more broadly applicable for enhancing the localization of bacteria to specified niches.

Injectable Therapeutic Organoids Using Sacrificial Hydrogels.

Organoids are becoming widespread in drug-screening technologies but have been used sparingly for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice, and rapidly formed perfusing vasculature. Moreover, in a mouse model of peripheral artery disease, intramuscular injections of blood-vessel units resulted in rapid restoration of vascular perfusion within seven days. As cell therapy transforms into a new class of therapeutic modality, this simple method-by making use of the dynamic nature of hydrogels-could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.

Rapid screening of engineered microbial therapies in a 3D multicellular model.

Synthetic biology is transforming therapeutic paradigms by engineering living cells and microbes to intelligently sense and respond to diseases including inflammation, infections, metabolic disorders, and cancer. However, the ability to rapidly engineer new therapies far outpaces the throughput of animal-based testing regimes, creating a major bottleneck for clinical translation. In vitro approaches to address this challenge have been limited in scalability and broad applicability. Here, we present a bacteria-in-spheroid coculture (BSCC) platform that simultaneously tests host species, therapeutic payloads, and synthetic gene circuits of engineered bacteria within multicellular spheroids over a timescale of weeks. Long-term monitoring of bacterial dynamics and disease progression enables quantitative comparison of critical therapeutic parameters such as efficacy and biocontainment. Specifically, we screen Salmonella typhimurium strains expressing and delivering a library of antitumor therapeutic molecules via several synthetic gene circuits. We identify candidates exhibiting significant tumor reduction and demonstrate high similarity in their efficacies, using a syngeneic mouse model. Last, we show that our platform can be expanded to dynamically profile diverse microbial species including Listeria monocytogenes, Proteus mirabilis, and Escherichia coli in various host cell types. This high-throughput framework may serve to accelerate synthetic biology for clinical applications and for understanding the host-microbe interactions in disease sites.

Engineering bacteria for cancer therapy.

The engineering of living cells and microbes is ushering in a new era of cancer therapy. Due to recent microbiome studies indicating the prevalence of bacteria within the human body and specifically in tumor tissue, bacteria have generated significant interest as potential targets for cancer therapy. Notably, a multitude of empirical studies over the past decades have demonstrated that administered bacteria home and grow in tumors due to reduced immune surveillance of tumor necrotic cores. Given their specificity for tumors, bacteria present a unique opportunity to be engineered as intelligent delivery vehicles for cancer therapy with synthetic biology techniques. In this review, we discuss the history, current state, and future challenges associated with using bacteria as a cancer therapy.

Outcomes of Infants Receiving Palivizumab Prophylaxis for Respiratory Syncytial Virus in Canada and Italy: An International, Prospective Cohort Study.

BACKGROUND: Respiratory syncytial virus (RSV) infection frequently results in RSV-related hospitalization (RSVH) in young infants. We examined the outcomes of palivizumab recipients within the Canadian Registry (CARESS) and the Torino-Verona Italian Registry over the 2002-2014 RSV seasons. METHODS: RSVHs were captured during the study seasons. Premature infants who received palivizumab (≤35 completed weeks' gestational age; group1) were compared with infants given palivizumab for underlying disorders regardless of gestational age (group 2). Variables and between-group incidences were analyzed. Risk factors associated with RSVH were assessed by logistic regression. RESULTS: A total of 14,468 palivizumab-exposed infants were enrolled (group 1, n = 9093; group 2, n = 4856; miscellaneous, n = 519). RSVH was significantly more frequent in group 2 (211/4856, 4.34%) versus group 1 infants (216/9093, 2.37% [relative risk 1.93; 95% confidence interval (CI): 1.60-2.33; P < 0.0001]). Infants with neuromuscular disorders (7.88%), airway anomalies (5.95%), bronchopulmonary dysplasia (4.75%) and hemodynamically significant congenital heart disease (4.10%) had the highest RSVH incidences. After multivariable logistic regression, only neuromuscular disease [odds ratio [OR] 4.29; 95% CI: 2.30-8.00; P < 0.01], airway anomalies (OR 3.23; 95% CI: 1.92-5.43; P < 0.01), Down syndrome (OR 2.25; 95% CI: 1.31-3.89; P < 0.01), hemodynamically significant congenital heart disease (OR 2.24; 95% CI: 1.52-3.31; P < 0.001), prematurity ≤28 completed weeks' gestational age (OR 1.82; 95% CI: 1.29-2.58; P < 0.001) and bronchopulmonary dysplasia (OR 1.81; 95% CI: 1.31-2.50; P < 0.001) significantly predicted RSVH. No significant association was detected with the number of doses administered or the time elapsed after the previous dose. CONCLUSIONS: RSVH rates are higher in infants given palivizumab for reasons other than prematurity. It is uncertain whether these findings relate to inadequate current palivizumab dosing protocols or to a specific increased RSVH risk inherent in infants with severe underlying comorbidities.

Risk Factors for Progression of Alzheimer Disease in a Canadian Population: The Canadian Outcomes Study in Dementia (COSID).

OBJECTIVE: To determine risk factors for clinically significant progression during 12 months in patients with mild-to-moderate Alzheimer disease. METHOD: Community-dwelling patients with mild-to-moderate Alzheimer disease were enrolled in a 3-year prospective study, the Canadian Outcomes Study in Dementia (commonly referred to as COSID), at 32 Canadian sites. Assessments included the Global Deterioration Scale (GDS) for disease severity, the Mini-Mental State Examination (MMSE) for cognition, the Functional Autonomy Measurement System (SMAF) for daily functioning, and the NeuroPsychiatric Inventory (NPI) for behaviour, measured at baseline and at 12 months. Logistic regression identified factors associated with GDS decline, and subsequent stepwise regression identified key independent predictors. Area under the curve (AUC) was then calculated for the model. RESULTS: Among 488 patients (mean age 76.5 years [SD 6.4], MMSE 22.1 [SD4.6], 44.1% male), 225 (46%) showed GDS decline. After adjusting for age, baseline risk factors for deterioration included the following: poorer cognition (lower MMSE score, OR 0.55; 95% CI 0.4 to 0.72 per 5 points, P ≤ 0.001), greater dependence (lower SMAF, OR 0.72; 95% CI 0.63 to 0.83 per 5 points, P ≤ 0.001), and more neuropsychiatric symptoms (higher NPI, OR 1.11; 95% CI 1.02 to 1.2 per 5 points, P = 0.02), with a protective effect of male sex (OR 0.59; 95% CI 0.39 to 0.9, P = 0.02), and higher (worse) GDS score (very mild, compared with mild OR 0.25; 95% CI 0.09 to 0.70, P ≤ 0.01; compared with moderate, OR 0.08; 95% CI 0.03 to 0.23, P < 0.001; compared with moderately severe, OR 0.03; 95% CI 0.01 to 0.11, P < 0.001). The AUC was 73% (P < 0.001) (sensitivity 90% and specificity 33%). CONCLUSION: The progression of Alzheimer disease in Canada can be predicted using readily available clinical information.

Predictors of readmission to a psychiatry inpatient unit.

OBJECTIVE: To determine predictors of time to readmission to a general psychiatry inpatient unit. METHOD: Data from the Minimum Data Set-Mental Health (MDS-MH), a standardized assessment used to collect demographic and clinical information, were retrospectively reviewed from April 2006 through October 2008. A total of 758 patients were eligible for the study. A set of clinically relevant predictors was generated based on a literature review. A Cox regression model was applied to determine which variables were most predictive of shorter time to readmission, and their respective hazard ratios (HR). RESULTS: Covariates that were significantly associated with readmission (HR [95% CI]) included receiving a pass (3.48 [2.33, 5.17], p ≤ 0.0005), 1-2 psychiatric admissions in the past two years (15.63 [7.50, 32.55], p ≤ 0.0005), and more than 3 psychiatric admissions in the past two years (24.15 [11.58, 50.36], p ≤ 0.0005). Post hoc analysis indicated that those issued passes were more commonly male (57.1% vs. 43.9%, p=0.03), with a longer length of stay (25.4 ± 21.2 days vs. 18.7 ± 21.1 days, p=0.008), and higher GAF score (62.8 ± 11.1 vs. 57.8 ± 13.9, p=0.003), but were otherwise similar. CONCLUSIONS: The factors that were associated with reduced time to readmission were a history of previous admissions and receipt of a pass prior to discharge. These results suggest that while physicians may be able to identify patients at high risk of early readmission, issuing a pass may not fully mitigate this risk. There is a need for critical research evaluating the potential benefits of passes.

Respiratory-related hospitalizations following prophylaxis in the Canadian registry for palivizumab (2005-2012) compared to other international registries.

Respiratory syncytial virus (RSV) infection occurs commonly in infants aged ≤2 years, and severe infection results in hospitalization with accompanying morbidity and mortality. Palivizumab has been available for prophylaxis for the past 15 years. Prospective data on patients who received palivizumab from 2005 to 2012 has been assembled in the Canadian registry (CARESS) to document utilization, compliance, and health outcomes in both hospital and community settings. Long-term data is necessary to evaluate the impact of palivizumab on the incidence of RSV infections, minimize healthcare resources, and identify which infant subpopulations are receiving prophylaxis. A database search was also conducted for similar information from published registries, and hospitalization rates were compared to results from randomized clinical trials (RCTs).Overall hospitalization rates (percent; range) for respiratory-related illnesses and RSV-specific infection in infants who meet standard indications for prophylaxis were 6.6 (3.3-7.7) and 1.55 (0.3-2.06), respectively, in CARESS, which closely aligns with registry data from 4 other countries, despite the former comprising the largest cohort of complex patients internationally. Overall RSV-related hospitalization rates were lower across registries compared to equivalent patients in RCTs. Registry data provides valuable information regarding real-world experience with palivizumab, while facilitating the genesis of new research themes.

Zinc in depression: a meta-analysis.

BACKGROUND: Zinc is an essential micronutrient with diverse biological roles in cell growth, apoptosis and metabolism, and in the regulation of endocrine, immune, and neuronal functions implicated in the pathophysiology of depression. This study sought to quantitatively summarize the clinical data comparing peripheral blood zinc concentrations between depressed and nondepressed subjects. METHODS: PubMed, Cumulated Index to Nursing and Allied Health Literature, and PsycINFO were searched for original peer-reviewed studies (to June 2012) measuring zinc concentrations in serum or plasma from depressed subjects (identified by either screening or clinical criteria) and nondepressed control subjects. Mean (±SD) zinc concentrations were extracted, combined quantitatively in random-effects meta-analysis, and summarized as a weighted mean difference (WMD). RESULTS: Seventeen studies, measuring peripheral blood zinc concentrations in 1643 depressed and 804 control subjects, were included. Zinc concentrations were approximately -1.85 µmol/L lower in depressed subjects than control subjects (95% confidence interval: [CI]: -2.51 to -1.19 µmol/L, Z17 = 5.45, p < .00001). Heterogeneity was detected (χ(2)17 = 142.81, p < .00001, I(2) = 88%) and explored; in studies that quantified depressive symptoms, greater depression severity was associated with greater relative zinc deficiency (B = -1.503, t9 = -2.82, p = .026). Effect sizes were numerically larger in studies of inpatients (WMD -2.543, 95% CI: -3.522 to -1.564, Z9 = 5.09, p < .0001) versus community samples (WMD -.943, 95% CI: -1.563 to -.323, Z7 = 2.98, p = .003) and in studies of higher methodological quality (WMD -2.354, 95% CI: -2.901 to -1.807, Z7 = 8.43, p < .0001). CONCLUSIONS: Depression is associated with a lower concentration of zinc in peripheral blood. The pathophysiological relationships between zinc status and depression, and the potential benefits of zinc supplementation in depressed patients, warrant further investigation.

Enlarged gold-tipped silicon microprobe arrays and signal compensation for multi-site electroretinogram recordings in the isolated carp retina.

In order to record multi-site electroretinogram (ERG) responses in isolated carp retinae, we utilized three-dimensional (3D), extracellular, 3.5-µm-diameter silicon (Si) probe arrays fabricated by the selective vapor-liquid-solid (VLS) growth method. Neural recordings with the Si microprobe exhibit low signal-to-noise (S/N) ratios of recorded responses due to the high-electrical-impedance characteristics of the small recording area at the probe tip. To increase the S/N ratio, we designed and fabricated enlarged gold (Au) tipped Si microprobes (10-µm-diameter Au tip and 3.5-µm-diameter probe body). In addition, we demonstrated that the signal attenuation and phase delay of ERG responses recorded via the Si probe can be compensated by the inverse filtering method. We conclude that the reduction of probe impedance and the compensation of recorded signals are useful approaches to obtain distortion-free recording of neural signals with high-impedance microelectrodes.

Electrical interfacing between neurons and electronics via vertically integrated sub-4 microm-diameter silicon probe arrays fabricated by vapor-liquid-solid growth.

We report here a technique for use in electrical interfaces between neurons and microelectronics, using vertically integrated silicon probe arrays with diameters of 2-3.5 microm and lengths of 60-120 microm. Silicon probe arrays can be fabricated by selective vapor-liquid-solid (VLS) growth. A doped n-type silicon probe with the resistance of 1 k Omega has an electrical impedance of less than 10 M Omega in physiological saline. After inserting the probe arrays into the retina of a carp (Cyrpinus carpio), we conducted electrical recording of neural signals, using the probes to measure light-evoked electrical neural signals. We determined that recorded signals represented local field potentials of the retina (electroretinogram (ERG)). The VLS-probe can provide minimally invasive neural recording/stimulation capabilities at high spatial resolution for fundamental studies of nervous systems. In addition, the probe arrays can be integrated with microelectronics; therefore, these probes make it possible to construct interfaces between neurons and microelectronics in advanced neuroscience applications.