The state of technological advancement to address challenges in the manufacture of rAAV gene therapies.

Current processes for the production of recombinant adeno-associated virus (rAAV) are inadequate to meet the surging demand for rAAV-based gene therapies. This article reviews recent advances that hold the potential to address current limitations in rAAV manufacturing. A multidisciplinary perspective on technological progress in rAAV production is presented, underscoring the necessity to move beyond incremental refinements and adopt a holistic strategy to address existing challenges. Since several recent reviews have thoroughly covered advancements in upstream technology, this article provides only a concise overview of these developments before moving to pivotal areas of rAAV manufacturing not well covered in other reviews, including analytical technologies for rapid and high-throughput measurement of rAAV quality attributes, mathematical modeling for platform and process optimization, and downstream approaches to maximize efficiency and rAAV yield. Novel technologies that have the potential to address the current gaps in rAAV manufacturing are highlighted. Implementation challenges and future research directions are critically discussed.

Cell size, density, and nutrient dependency of unicellular algal gravitational sinking velocities.

Eukaryotic phytoplankton, also known as algae, form the basis of marine food webs and drive marine carbon sequestration. Algae must regulate their motility and gravitational sinking to balance access to light at the surface and nutrients in deeper layers. However, the regulation of gravitational sinking remains largely unknown, especially in motile species. Here, we quantify gravitational sinking velocities according to Stokes' law in diverse clades of unicellular marine microalgae to reveal the cell size, density, and nutrient dependency of sinking velocities. We identify a motile algal species, Tetraselmis sp., that sinks faster when starved due to a photosynthesis-driven accumulation of carbohydrates and a loss of intracellular water, both of which increase cell density. Moreover, the regulation of cell sinking velocities is connected to proliferation and can respond to multiple nutrients. Overall, our work elucidates how cell size and density respond to environmental conditions to drive the vertical migration of motile algae.

Constant surface area-to-volume ratio during cell growth as a design principle in mammalian cells.

All cells are subject to geometric constraints, such as surface area-to-volume (SA/V) ratio, that impact cell functions and force biological adaptations. Like the SA/V ratio of a sphere, it is generally assumed that the SA/V ratio of cells decreases as cell size increases. Here, we investigate this in near-spherical mammalian cells using single-cell measurements of cell mass and surface proteins, as well as imaging of plasma membrane morphology. We find that the SA/V ratio remains surprisingly constant as cells grow larger. This observation is largely independent of the cell cycle and the amount of cell growth. Consequently, cell growth results in increased plasma membrane folding, which simplifies cellular design by ensuring sufficient membrane area for cell division, nutrient uptake and deformation at all cell sizes.

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease.

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples.

Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples.

Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and therefore a fundamental determinant of cell state and function. However, existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples. Here we present an approach for measuring the density of 30,000 single cells per hour with a precision of 0.03% (0.0003 g/mL) by integrating fluorescence exclusion microscopy with a suspended microchannel resonator. Applying this approach to human lymphocytes, we discovered that cell density and its variation decrease as cells transition from quiescence to a proliferative state, suggesting that the level of molecular crowding decreases and becomes more regulated upon entry into the cell cycle. Using a pancreatic cancer patient-derived xenograft model, we found that the ex vivo density response of primary tumor cells to drug treatment can predict in vivo tumor growth response. Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a new biomarker for functional precision medicine.

Leukemia circulation kinetics revealed through blood exchange method.

Leukemias and their bone marrow microenvironments undergo dynamic changes over the course of disease. However, little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of CLC dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: (i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and (ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

Single-cell mass distributions reveal simple rules for achieving steady-state growth.

IMPORTANCE: Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

Leukemia circulation kinetics revealed through blood exchange method.

Leukemias and their bone marrow microenvironment are known to undergo dynamic changes over the course of disease. However, relatively little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of leukemia cell dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

Nucleotide imbalance decouples cell growth from cell proliferation.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.

Single-cell monitoring of dry mass and dry mass density reveals exocytosis of cellular dry contents in mitosis.

Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

Weighing the DNA Content of Adeno-Associated Virus Vectors with Zeptogram Precision Using Nanomechanical Resonators.

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-associated virus (AAV) vectors, which are the most widely used viral vectors for in vivo gene therapy, are typically characterized using PCR, ELISA, and analytical ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as charge-detection mass spectroscopy, static light scattering, and mass photometry offer turnaround times of minutes for measuring AAV mass using optical or charge properties of AAV. Here, we demonstrate an orthogonal method where suspended nanomechanical resonators (SNR) are used to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a precision near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Cell size is a determinant of stem cell potential during aging.

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

Functional drug susceptibility testing using single-cell mass predicts treatment outcome in patient-derived cancer neurosphere models.

Functional precision medicine aims to match individual cancer patients to optimal treatment through ex vivo drug susceptibility testing on patient-derived cells. However, few functional diagnostic assays have been validated against patient outcomes at scale because of limitations of such assays. Here, we describe a high-throughput assay that detects subtle changes in the mass of individual drug-treated cancer cells as a surrogate biomarker for patient treatment response. To validate this approach, we determined ex vivo response to temozolomide in a retrospective cohort of 69 glioblastoma patient-derived neurosphere models with matched patient survival and genomics. Temozolomide-induced changes in cell mass distributions predict patient overall survival similarly to O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and may aid in predictions in gliomas with mismatch-repair variants of unknown significance, where MGMT is not predictive. Our findings suggest cell mass is a promising functional biomarker for cancers and drugs that lack genomic biomarkers.

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice.

Existing preclinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1-2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

Oncogenic HSP90 Facilitates Metabolic Alterations in Aggressive B-cell Lymphomas.

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane-bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors. SIGNIFICANCE: The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.

Inertial and viscous flywheel sensing of nanoparticles.

Rotational dynamics often challenge physical intuition while enabling unique realizations, from the rotor of a gyroscope that maintains its orientation regardless of the outer gimbals, to a tennis racket that rotates around its handle when tossed face-up in the air. In the context of inertial sensing, which can measure mass with atomic precision, rotational dynamics are normally considered a complication hindering measurement interpretation. Here, we exploit the rotational dynamics of a microfluidic device to develop a modality in inertial sensing. Combining theory with experiments, we show that this modality measures the volume of a rigid particle while normally being insensitive to its density. Paradoxically, particle density only emerges when fluid viscosity becomes dominant over inertia. We explain this paradox via a viscosity-driven, hydrodynamic coupling between the fluid and the particle that activates the rotational inertia of the particle, converting it into a 'viscous flywheel'. This modality now enables the simultaneous measurement of particle volume and mass in fluid, using a single, high-throughput measurement.

Variations of intracellular density during the cell cycle arise from tip-growth regulation in fission yeast.

Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.