Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Engineered cell entry links receptor biology with single-cell genomics.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

Designer Assays for Your Sick, Subdivided Heart.

Using induced pluripotent stem cells and microelectromechanical device technology Zhao et al. have developed 'organs on chips' representing the different chambers of the heart and used them to replicate healthy and diseased tissues in vitro. These systems offer investigators and the pharmaceutical industry a new tool in testing the safety and efficacy of new medicinal therapeutics.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Mouse brain elastography changes with sleep/wake cycles, aging, and Alzheimer's disease.

Understanding the physiological processes in aging and how neurodegenerative disorders affect cognitive function is a high priority for advancing human health. One specific area of recently enabled research is the in vivo biomechanical state of the brain. This study utilized reverberant optical coherence elastography, a high-resolution elasticity imaging method, to investigate stiffness changes during the sleep/wake cycle, aging, and Alzheimer's disease in murine models. Four-dimensional scans of 44 wildtype mice, 13 mice with deletion of aquaporin-4 water channel, and 12 mice with Alzheimer-related pathology (APP/PS1) demonstrated that (1) cortical tissue became softer (on the order of a 10% decrease in shear wave speed) when young wildtype mice transitioned from wake to anesthetized, yet this effect was lost in aging and with mice overexpressing amyloid-ß or lacking the water channel AQP4. (2) Cortical stiffness increased with age in all mice lines, but wildtype mice exhibited the most prominent changes as a function of aging. The study provides novel insight into the brain's biomechanics, the constraints of fluid flow, and how the state of brain activity affects basic properties of cortical tissues.

Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles.

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

Cellular and Engineered Organoids for Cardiovascular Models.

An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight future directions.

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Breast Ultrasound Volume Sweep Imaging: A New Horizon in Expanding Imaging Access for Breast Cancer Detection.

OBJECTIVE: The majority of people in the world lack basic access to breast diagnostic imaging resulting in delay to diagnosis of breast cancer. In this study, we tested a volume sweep imaging (VSI) ultrasound protocol for evaluation of palpable breast lumps that can be performed by operators after minimal training without prior ultrasound experience as a means to increase accessibility to breast ultrasound. METHODS: Medical students without prior ultrasound experience were trained for less than 2 hours on the VSI breast ultrasound protocol. Patients presenting with palpable breast lumps for standard of care ultrasound examination were scanned by a trained medical student with the VSI protocol using a Butterfly iQ handheld ultrasound probe. Video clips of the VSI scan imaging were later interpreted by an attending breast imager. Results of VSI scan interpretation were compared to the same-day standard of care ultrasound examination. RESULTS: Medical students scanned 170 palpable lumps with the VSI protocol. There was 97% sensitivity and 100% specificity for a breast mass on VSI corresponding to 97.6% agreement with standard of care (Cohen's κ = 0.95, P < .0001). There was a detection rate of 100% for all cancer presenting as a sonographic mass. High agreement for mass characteristics between VSI and standard of care was observed, including 87% agreement on Breast Imaging-Reporting and Data System assessments (Cohen's κ = 0.82, P < .0001). CONCLUSIONS: Breast ultrasound VSI for palpable lumps offers a promising means to increase access to diagnostic imaging in underserved areas. This approach could decrease delay to diagnosis for breast cancer, potentially improving morbidity and mortality.

RNA-GPS predicts high-resolution RNA subcellular localization and highlights the role of splicing.

Subcellular localization is essential to RNA biogenesis, processing, and function across the gene expression life cycle. However, the specific nucleotide sequence motifs that direct RNA localization are incompletely understood. Fortunately, new sequencing technologies have provided transcriptome-wide atlases of RNA localization, creating an opportunity to leverage computational modeling. Here we present RNA-GPS, a new machine learning model that uses nucleotide-level features to predict RNA localization across eight different subcellular locations-the first to provide such a wide range of predictions. RNA-GPS's design enables high-throughput sequence ablation and feature importance analyses to probe the sequence motifs that drive localization prediction. We find localization informative motifs to be concentrated on 3'-UTRs and scattered along the coding sequence, and motifs related to splicing to be important drivers of predicted localization, even for cytotopic distinctions for membraneless bodies within the nucleus or for organelles within the cytoplasm. Overall, our results suggest transcript splicing is one of many elements influencing RNA subcellular localization.

A bioinspired and hierarchically structured shape-memory material.

Shape-memory polymeric materials lack long-range molecular order that enables more controlled and efficient actuation mechanisms. Here, we develop a hierarchical structured keratin-based system that has long-range molecular order and shape-memory properties in response to hydration. We explore the metastable reconfiguration of the keratin secondary structure, the transition from α-helix to ß-sheet, as an actuation mechanism to design a high-strength shape-memory material that is biocompatible and processable through fibre spinning and three-dimensional (3D) printing. We extract keratin protofibrils from animal hair and subject them to shear stress to induce their self-organization into a nematic phase, which recapitulates the native hierarchical organization of the protein. This self-assembly process can be tuned to create materials with desired anisotropic structuring and responsiveness. Our combination of bottom-up assembly and top-down manufacturing allows for the scalable fabrication of strong and hierarchically structured shape-memory fibres and 3D-printed scaffolds with potential applications in bioengineering and smart textiles.

Diagnostic Performance of an Artificial Intelligence System in Breast Ultrasound.

OBJECTIVES: We study the performance of an artificial intelligence (AI) program designed to assist radiologists in the diagnosis of breast cancer, relative to measures obtained from conventional readings by radiologists. METHODS: A total of 10 radiologists read a curated, anonymized group of 299 breast ultrasound images that contained at least one suspicious lesion and for which a final diagnosis was independently determined. Separately, the AI program was initialized by a lead radiologist and the computed results compared against those of the radiologists. RESULTS: The AI program's diagnoses of breast lesions had concordance with the 10 radiologists' readings across a number of BI-RADS descriptors. The sensitivity, specificity, and accuracy of the AI program's diagnosis of benign versus malignant was above 0.8, in agreement with the highest performing radiologists and commensurate with recent studies. CONCLUSION: The trained AI program can contribute to accuracy of breast cancer diagnoses with ultrasound.

Inhibition of mTOR Signaling Enhances Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells via p53-Induced Quiescence.

BACKGROUND: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)-signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. METHODS: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. RESULTS: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. CONCLUSIONS: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

Capturing the dynamics of small populations: A retrospective assessment using long-term data for an island reintroduction.

The art of population modelling is to incorporate factors essential for capturing a population's dynamics while otherwise keeping the model as simple as possible. However, it is unclear how optimal model complexity should be assessed, and whether this optimal complexity has been affected by recent advances in modelling methodology. This issue is particularly relevant to small populations because they are subject to complex dynamics but inferences about those dynamics are often constrained by small sample sizes. We fitted Bayesian hierarchical models to long-term data on vital rates (survival and reproduction) for the toutouwai Petroica longipes population reintroduced to Tiritiri Matangi, a 220-ha New Zealand island, and quantified the performance of those models in terms of their likelihood of replicating the observed population dynamics. These dynamics consisted of overall growth from 33 (±0.3) to 160 (±6) birds from 1992-2018, including recoveries following five harvest events for further reintroductions to other sites. We initially included all factors found to affect vital rates, which included inbreeding, post-release effects (PRE), density-dependence, sex, age and random annual variation, then progressively removed these factors. We also compared performance of models where data analysis and simulations were done simultaneously to those produced with the traditional two-step approach, where vital rates are estimated first then fed into a separate simulation model. Parametric uncertainty and demographic stochasticity were incorporated in all projections. The essential factors for replicating the population's dynamics were density-dependence in juvenile survival and PRE, i.e. initial depression of survival and reproduction in translocated birds. Inclusion of other factors reduced the precision of projections, and therefore the likelihood of matching observed dynamics. However, this reduction was modest when the modelling was done in an integrated framework. In contrast, projections were much less precise when done with a two-step modelling approach, and the cost of additional parameters was much higher under the two-step approach. These results suggest that minimization of complexity may be less important than accounting for covariances in parameter estimates, which is facilitated by integrating data analysis and population projections using Bayesian methods.

Applying a values-based decision process to facilitate comanagement of threatened species in Aotearoa New Zealand.

Ko koe ki tena, ko ahau ki tenai kiwai o te kete (you at that, and I at this handle of the basket). This Maori (New Zealanders of indigenous descent) saying conveys the principle of cooperation-we achieve more through working together, rather than separately. Despite decades of calls to rectify cultural imbalance in conservation, threatened species management still relies overwhelmingly on ideas from Western science and on top-down implementation. Values-based approaches to decision making can be used to integrate indigenous peoples' values into species conservation in a more meaningful way. We used such a values-based method, structured decision making, to develop comanagement of pekapeka (Mystacina tuberculata) (short-tailed bat) and tara iti (Sternula nereis davisae) (Fairy Tern) between Maori and Pakeha (New Zealanders of European descent). We implemented this framework in a series of workshops in which facilitated discussions were used to gather expert knowledge to predict outcomes and make management recommendations. For both species, stakeholders clearly stated their values as fundamental objectives from the start, which allowed alternative strategies to be devised that naturally addressed their diverse values, including matauranga Maori (Maori knowledge and perspectives). On this shared basis, all partners willingly engaged in the process, and decisions were largely agreed to by all. Most expectations of conflicts between values of Western science and Maori culture were unfounded. Where required, positive compromises were made by jointly developing alternative strategies. The values-based process successfully taha wairua taha tangata (brought both worlds together to achieve the objective) through codeveloped recovery strategies. This approach challenges the traditional model of scientists first preparing management plans focused on biological objectives, then consulting indigenous groups for approval. We recommend values-based approaches, such as structured decision making, as powerful methods for development of comanagement conservation plans between different peoples.

Aplicación de un Proceso de Decisiones Basadas en Valores para Facilitar el Comanejo de Especies Amenazadas en Aotearoa Nueva Zelanda Resumen Ko koe ki tena, ko ahau ki tenai kiwai o te kete (tú en ésa y yo en esta asa de la cesta). Este dicho Maori (neozelandeses con ascendencia indígena) expresa el principio de la cooperación - logramos más trabajando juntos que por separado. A pesar de las décadas de peticiones para rectificar el desbalance ambiental que existe en la conservación, el manejo de especies amenazadas todavía depende abrumadoramente de ideas tomadas de la ciencia occidental y en la implementación de arriba-abajo. Los enfoques para la toma de decisiones basados en valores pueden usarse para integrar de manera más significativa los valores de los pueblos indígenas dentro de la conservación de especies. Usamos un método basado en valores, la toma estructurada de decisiones, para desarrollar una estrategia de comanejo del pekapeka (Mystacina tuberculata) (murciélago de cola corta) y el tara iti (Sternula nereis davisae) (charrancito australiano) entre los Maori y los Pakeha (neozelandeses de ascendencia europea). Implementamos este marco de trabajo en una serie de talleres en los cuales se usaron discusiones facilitadas para recabar el conocimiento de los expertos para pronosticar los resultados y realizar recomendaciones de manejo. Para ambas especies, los actores sociales mencionaron claramente a sus valores como objetivos fundamentales desde el inicio, lo que permitió el diseño de estrategias alternativas que consideraran naturalmente estos diferentes valores, incluyendo el matauranga Maori (conocimiento y perspectivas Maori). Sobre esta base compartida, todos los colaboradores participaron voluntariamente en el proceso y la mayoría estuvo de acuerdo con las decisiones. La mayoría de los conflictos esperados entre la ciencia occidental y la cultura Maori no tuvieron fundamentos. En donde fueron requeridos, se realizaron concesiones positivas mediante el desarrollo conjunto de estrategias alternativas. El proceso basado en valores logró exitosamente taha wairua taha tangata (juntó a ambos mundos para conseguir el objetivo) por medio de estrategias de recuperación desarrolladas en conjunto. Esta estrategia desafía el modelo tradicional de los científicos preparando primero los planes de manejo enfocados en objetivos biológicos para después consultar a los grupos indígenas para que los aprueben. Recomendamos estos enfoques basados en valores, como la toma estructurada de decisiones, como métodos poderosos para el desarrollo de planes de conservación que incluyan el comanejo entre diferentes pueblos y personas.