UCSF

Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

The purpose of this study was to compare skeletal and dentoalveolar shape and size differences in patients with maxillary impacted canines versus unaffected patients using landmarked CBCT images and geometric morphometrics.104 cone-beam computed tomography scans from patients presenting with palatal or buccal impacted maxillary canines and 40 control patients with no impacted dentition were landmarked. 146 landmarks were plotted in Stratovan Checkpoint. Cone-beam computed tomography images including the nasal cavity, palate, sinus, alveolar crest, maxillary lateral walls, and dentition were landmarked by five examiners. No patient had undergone any type of orthodontic treatment in the past. Landmarked CBCT images were evaluated for shape and symmetry using geometric morphometric analysis by performing Procrustes superimposition, principal component analysis, and canonical variates analysis. Shape differences were further investigated by using linear regression analyses. Size differences were then investigated by looking at centroid size based on ratios between dentition and the entire landmarked maxilla. To analyze shape differences, we utilized a principal components analysis and found impaction location of the canine contributed to the primary and secondary principal component of the variance in the shape. Removing the variance of tooth location, a principal component analysis was conduct on the asymmetric component. We found patients with maxillary canine impactions had a constriction of the arch on the side of the impaction as well as lowering of the sinus floor on the side of the maxillary canine impaction compared to the control. The shape differences found were not statistically significant and we found no difference in shape between palatal versus buccal impactions. Looking at size with differences of the dentition, the centroid size of the maxillary canine, central incisor and first molar were statistically different between the impacted and nonimpacted groups with a reduction in centroid size of the canine in the impacted versus nonimpacted groups. There were no statistically significant size differences between the impacted maxillary canine groups however there was a reduction in size in the palatal size versus the buccal side. The centroid sizes of the maxilla without teeth, arch width, and palate size in the impacted versus the nonimpacted group were all statistically different from the control group with the impacted group being smaller while there was no difference between the palatal versus buccal groups. When looking at the size of the maxilla between males versus females, it was found that males consistently had large centroid sizes in their overall maxilla compared to females. We isolating for gender, there was no statistically significant difference in maxilla size between maxillary impaction palatal or buccal impactions. Patients with impacted maxillary canines have both shape and size differences in the overall maxillary structure as well as the maxillary canine compared to patients without impacted maxillary canines. Amongst the dentition there was a general size reduction between the impacted versus nonimpacted tooth sizes. The impacted canines and first incisors and first molars on the side of the impaction had a statistically significant reduction in size compared to the nonimpacted canine patients. We also found a reduction in overall maxillary size in patients with impacted canines. There was also no statistically significant difference in centroid size of teeth or maxillary structures between palatal and buccal groups. These findings reflect that perhaps the etiology of canine impaction location is in line with the genetic theory rather than a lack of guidance from an undersized lateral incisor.

Tumor progression is the major cause of death in cancer patients. Due to their higher chromosomal instability and other genomic alterations, tumors evolve rapidly in response to therapeutic interventions and other external pressures. The immune system is our first line of defense against cancer and its interaction with cancer cells can both constrain and promote tumor growth and metastasis. A heterogeneous tumor consists of subclones with different characteristics. During tumor progression the anti-tumor immune activity removes subclones that express highly immunogenic antigens and leaves behind cancer cells with high immune escape or immunosuppressive properties. This process is called tumor immunoediting. Studying tumor progression requires reliable in vivo models that effectively capture the intricacies and complexities of this process. During the 1970s, Isaiah Fidler demonstrated that repeated passaging of cancer cells in mice can be used to emulate metastatic progression. This in vivo selection model has been used by many different research groups (including us) to model tumor progression in a number of cancer models. Our group has utilized these in vivo selection models to study cell autonomous mechanisms of tumor progression. More recently, however, we have come to realize that by leveraging these in vivo-selection models we can focus on studying non-cell autonomous mechanisms. Building on this notion, here, we propose a generalization of in vivo selection that models the role of the immune system in shaping tumor evolution. Our “immune selection” model takes advantage of a panel of genetic mouse models with various degrees of immunocompetency to serve as hosts for established syngeneic tumor cell lines. We utilized these ‘immuno-selected’ derivatives, in conjunction with cutting-edge tools in genetic engineering and single-cell genomics, to study the tumor-immune co-evolution. We discovered that the interferon response pathway lies at the heart of tumor immune evasion. Additionally, we have uncovered novel molecular pathways responsible for conferring resistance to both antitumor immunity and immunotherapies. Targeting these pathways holds significant therapeutic potential, particularly when used in conjunction with immune checkpoint blockades (ICBs) and other forms of immunotherapy. The second part of this thesis is focused on utilizing machine learning and computational tools to identify important molecular mechanisms in small RNA secretion. We developed ExoGRU, a deep-learning model for predicting secretion probabilities of small RNAs based on their primary sequence. We used ExoGRU to (i) identify mutations that abrogate the secretion of known cell-free small RNAs, and (ii) predict high confidence sets of synthetic sequences that are secreted or retained. We also used independent experimental approaches to validate our model’s prediction abilities. We discovered that the molecular signature needed for small RNA secretion lies in its primary sequence. Furthermore, we identified both previously known and novel RNA binding proteins (RBPs) crucial for facilitating this secretion. In both projects discussed, we demonstrate the effectiveness of in vivo, high-throughput, multi-omics and computational tools in uncovering novel mechanisms, particularly in the evolution of tumor immunity and RNA secretion, areas traditionally challenging to explore with conventional methods.

Our behaviors during social interactions are wide-ranging and highly contextual. The norms that dictate how we should act vary based on who we are interacting with, societal context, and past experiences. Social cognition refers to the ability of animals to utilize both experience and the knowledge of learned social norms to select appropriate behaviors for specific contexts. The medial prefrontal cortex (mPFC) has a long-established role in executive function and cognition, and has also been found to be critical for the production of normal social behavior. However, the precise role of the mPFC in social behavior and the neural dynamics that occur in this region during social interaction have not been explored in great detail. Elucidating the precise role of the mPFC during social interaction is critical to understanding how abnormal social phenotypes arise in the array of psychiatric illnesses that impinge upon the PFC. One way to approach this problem is to utilize rodent models in combination with the wide array of molecular and genetic tools available in this system. These tools allow for studying the role of particular brain regions, neuron subtypes, and genes in behavior. Particularly, one tool that has expanded our understanding of the neural foundations of complex behavior is in vivo calcium imaging of neuronal activity with miniaturized, head-mounted microscopes. In combination with tools that allow for expression of the genetically encoded calcium indicator GCaMP in genetically-defined neuron subpopulations, this approach makes it possible to record activity specifically from cell types that have relevance to psychiatric disorders while animals can move and behave freely. Here, we describe two distinct studies that utilize this approach to record activity in the mPFC of mice from neuron populations that have been implicated in psychiatric disorders. In the first study, we record activity in layer 5 projection neurons of the mPFC in both wild-type mice and mice that have the Tbr1 gene deleted specifically in cortical layer 5 neurons. Tbr1 is a high-confidence autism risk-gene, and mPFC layer 5 projection neurons have been identified as a hub of autism risk-gene expression. We find that these Tbr1 layer 5 conditional knockout animals (Tbr1-cKO) display abnormal social and anxiety-related avoidance behaviors. During social interactions, encoding of behavior by correlated activity of mPFC neurons is diminished in cKO animals, while correlated activity remains intact during anxiety-related behavior. We also identify signals in mPFC neural ensembles that are predictive of approach-avoidance decisions, but are lost in the Tbr1 cKO mouse model. In the second study, we record specifically from mPFC neurons that express the dopamine receptor D2R, which has been implicated in disorders including schizophrenia and depression. We also record from mPFC D2R+ neurons after knocking out the D2R, to assess the role of the receptor itself in socially recruited activity. These animals performed the 3-chamber social assay, and we find significant center chamber associated activity in D2R+ neurons that is lost when the D2R is deleted. Inhibition of mPFC D2R+ neurons specifically in the center chamber of this task leads to an overall increase in the number of social interactions.

To orchestrate the complex events of craniofacial morphogenesis requires a concerted effort between gene expression and tissue specific events. In the embryonic midface, facial prominences must execute a coordinated fusion between maxillary, medial and lateral nasal prominences at the lambdoidal junction, requiring breakdown and removal of epithelium to permit coalescence of underlying mesenchyme. When this process is disrupted by genetic mutations, cleft lip, the most common craniofacial birth defect in humans, is created. Key to understanding cleft lip pathogenesis is the reliance on mouse models. Models of cleft lip and studies of the role of epithelium are rare in comparison to cleft palate and research on underlying mesenchyme. The epithelium possesses discrete roles that facilitate fusion at the lambdoidal junction, including apoptosis and epithelial to mesenchymal transitions. To better understand its heterogeneity, we sought to enrich for the midface epithelium and isolate it from mesenchyme to gain fuller resolution of the lambdoidal junction transcriptome. This dissertation contains the product of that endeavor to focus on the role of the epithelium alone both in normal conditions and in cleft lip pathogenesis. In doing so, an atlas of lambdoidal junction epithelium at timepoints before, during and after fusion of the midface was created and validated. Through these methods, a deep analysis of each cluster resulted in the localization of a cluster undergoing cell cycle arrest and located at the fusion site of each prominence. A series of experiments were conducted which demonstrated that cell cycle arrest is characteristic of this population that is altered in cleft lip models. Additional research yielded ties to normal midface morphogenesis as well as novel orofacial cleft risk candidates, including Zfhx3 a gene with ties to cell cycle arrest. This thesis also expands upon the phenotypic characterization of a novel mouse cross which conditionally removes Bmpr1a from the ectoderm of the embryo, creating a model of cleft lip and palate. This work collectively produces knowledge of the midface epithelium at a scale of resolution not seen before as well as describes in detail how cell cycle arrest is a characteristic of primary palate fusion.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.

Background: The pregnancy desires of adolescents and young adults are not well understood, as researchers and health care providers have traditionally assumed that individuals in this age group want to prevent pregnancy. Efforts to promote reproductive autonomy and promote contraceptive use may have been misguided as they relied on research data based on this assumption. Traditional methods for assessing pregnancy intention use retrospective and binary measures that do not capture the range of feelings around pregnancy. Development of a new validated measure, the Desire to Avoid Pregnancy (DAP) scale, allows for a more holistic perspective, representing a range of pregnancy preferences. Methods: This dissertation study is a secondary analysis of data from the Attitudes and Decision After Pregnancy Testing (ADAPT) study, which recruited participants from March 2019 to October 2022. Using the subset of participants aged 15 to 24 years old at enrollment (N= 1,020), pregnancy preferences were measured with the DAP scale and various demographic, contextual, and economic participant characteristics were analyzed in relation to these preferences and contraceptive use. This dissertation research described the range of youth pregnancy preferences and investigated factors associated with these preferences. The degree to which pregnancy preferences are aligned with contraceptive use was studied and whether these preferences mediated the effect of contextual factors on contraceptive use. Subsequent analysis investigated participants whose pregnancy preferences did not align with their contraceptive use, to identify individuals who may be at risk of compromised reproductive autonomy. Results: Young people had a range of pregnancy preferences, including a high desire to avoid pregnancy, ambivalence, and a low desire to avoid pregnancy. Factors significantly associated with greater desire to avoid pregnancy were identifying as White (compared to Latine), having depressive symptoms, being enrolled in school, having a mother with higher educational attainment, and not having a main partner (compared to being in a high quality relationship). Factors significantly associated with more openness to pregnancy were having one child (compared to none) and being religious. Contraceptive use was more likely among youth who wanted to prevent pregnancy. Interestingly, both youth in high quality relationships (compared to no relationship) and religious youth were more open to pregnancy yet more likely to use contraceptives. As pregnancy preferences acted as a suppressing mediator for these two variables, the degree to which these participants are more likely to use contraception becomes more notable. At some points during the study period, participants’ pregnancy preferences did not align with contraceptive use and a few participants’ characteristics were associated with these discordant relationships. Contraceptive use declined by increasing age for both those who wanted to prevent and those open to pregnancy. Among those with a greater desire to avoid pregnancy, those not in school or not in a relationship were less likely to use a contraceptive. Among youth open to pregnancy, multiparous participants were more likely to use a contraceptive. Conclusion: Having a more nuanced understanding of how youth pregnancy preferences are impacted by contextual factors and how in turn these pregnancy preferences impact contraceptive use, can help to direct policy to help meet the reproductive needs of this population.

Timing mechanisms in biology remain poorly understood. As one prime example, little is known about the mechanisms that specify how long the gestating uterus will remain quiescent before entering labor. Our lack of insight into this fundamental question, which applies to all mammalian species, also limits investigation into potential causes of preterm labor, a major human pregnancy complication. My dissertation work provides evidence that gestation length in mice is determined by an epigenetic timer that runs autonomously within the fibroblasts of the pregnant uterus. The timer is set during the peri-implantation period when select loci establish appropriate levels of the repressive histone mark H3K27me3. These loci then progressively lose H3K27me3, thereby scheduling the uterine cell state transitions and associated gene expression changes of late gestation that are the proximal mediators of luteolysis (progesterone withdrawal) and labor onset. Initial overwinding of the timer via genetic ablation of the histone demethylase KDM6B delays these transitions and extends gestation length. My findings also demonstrate requirements for KDM6 demethylases in inflammation-induced preterm labor, and suggest potential requirements for KDM6B in the uterine-intrinsic pathways of parturition that are distinct from luteolysis. These results unexpectedly implicate epigenetic pathways in fibroblasts as a top-level determinant of both normal and pathological parturition mechanisms. We anticipate that further dissection of the ways such fibroblast programming controls gestation length may suggest novel approaches for improving human pregnancy outcomes.

The temporomandibular joint (TMJ) is a complex structure that joins the temporal bone to the mandible in order to facilitate mastication and speech. All the components of the TMJ must work in tandem with the contralateral side to produce the dynamic movements required for speech and mastication. Any dysfunction between the two TMJs or within one TMJ itself may lead to painful or non-painful temporomandibular disorders (TMDs). There is a wide range of causes for TMDs, including biomechanical, biological, and bio-psychosocial factors, but there are limited treatment options available due to a lack of understanding of the etiology and pathogenesis of TMDs. In order to provide improved interventions and treatment, there needs to be a better understanding of the etiopathogenesis of TMDs at the tissue, cellular, and molecular level. One specific area of interest is the mandibular condylar cartilage (MCC), which has been shown to undergo cellular changes in response to altered occlusion in mice. The aim of this study was to examine how the TMJ responds to altered occlusion (unilateral maxillary molar tooth extractions) in skeletally and dentally mature mice to better understand the adaptive potential of the TMJ and progression of occlusal-related TMDs. The results showed that there were no changes in morphology of the condyle or cellular organization of the MCC in adult mice after extraction of the 3 maxillary right molars, and thus, no adaptive changes occur in response to altered occlusion in skeletally mature mice with stable occlusion.

Nature challenges healthy mammals with constant risk of infection by a wide variety of pathogens and with degradation of healthy tissue and control systems. I have been interested in drug delivery and improved delivery of therapeutics primarily to attack pathogens but with incidental value in assessing vital functions of a healthy mammal. My recent work uses protein design to approach such problems. Delivering biomacromolecules remains important both for therapeutics and in discerning and shaping functions of cells.My first thesis project focused on designing a better glomerular filtration rate (GFR) marker to facilitate assessment of renal function and seeking a marker reflecting water distribution in the body, which is relevant to distribution of highly soluble drugs [1]. Tritiated polyethylene glycol (PEG) was known to clear the kidneys effectively and correlated well with a GFR standard assay. A better radioactive label for PEG would allow for easy detection, including imaging. At the time of the project, only one biological conjugation with PEG was reported. Attaching an iodinatable moiety to polyethylene glycol (PEG) polymers of different sizes enabled tracking the compound using radioactive iodine. I made a series of related compounds and studied the pharmacokinetics (PK) and pharmacodynamics (PD) of these in rodents and ultimately in a dog. Using relatively long PEG polymers of molecular weight (MW) 5,000 to 6,000 daltons, the PEG dominated the behavior of the compounds, clearing rapidly through the kidneys. With shorter PEG polymers, the chemistry of the iodinatable group was more significant and the compounds were more likely to clear through the bile, to a degree making them unsuitable for a GFR marker but possibly useful to study liver function. Chapter 1, the published manuscript from my first thesis project, is cited in 20 scientific publications and 51 issued US patents. Variations on the design principles of my project have been used widely in the pharmaceutical industry. During a break in my PhD studies, I improved the formulation of a human Phase-2-ready antifungal drug and designed and organized extensive testing in mice and dogs to show that a sustained-release formulation would overcome PK limitations and made the drug much more potent. This work is discussed briefly in Chapter 2. My second thesis project studied brilacidin activity against 40 fungal isolates from 20 different species, showing useful activity against several important human pathogens [2]. The human and many other innate immune systems includes a variety of peptides known as defensins that weaken or kill a variety of pathogens, including bacteria, fungi, and viruses. Brilacidin is a synthetic defensin-mimic, designed to exhibit the physicochemical properties of defensins as a class. Brilacidin is in human Phase 2 trials. Despite its potential, Brilacidin's efficacy against fungi had not been comprehensively explored until my studies, which showcased its viability as a therapeutic agent against challenging-to-treat fungal infections, thereby offering a beacon of hope for future clinical interventions. Chapter 3, the published manuscript based on this second thesis project, has recently been submitted for review, available online in Preprints.