Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy.

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.

Empirical evidence for biometal dysregulation in Parkinson's disease from a systematic review and Bradford Hill analysis.

The Bradford Hill model evaluates the causal inference of one variable on another by assessing whether evidence of the suspected causal variable aligns with a set of nine criteria proposed by Bradford Hill, each representing fundamental tenets of a causal relationship. The aim of this study was to use the Bradford Hill model of causation to assess the level of empirical evidence supporting our hypotheses that alterations to iron and copper levels, and iron- and copper-associated proteins and genes, contribute to Parkinson's disease etiology. We conducted a systematic review of all available articles published to September 2019 in four online databases. 8437 articles matching search criteria were screened for pre-defined inclusion and exclusion criteria. 181 studies met study criteria and were subsequently evaluated for study quality using established quality assessment tools. Studies meeting criteria for moderate to high quality of study design (n = 155) were analyzed according to the Bradford Hill model of causation. Evidence from studies considered of high quality (n = 73) supported a causal role for iron dysregulation in Parkinson's disease. A causal role for copper dysregulation in Parkinson's disease was also supported by high quality studies, although substantially fewer studies investigated copper in this disorder (n = 25) compared with iron. The available evidence supports an etiological role for iron and copper dysregulation in Parkinson's disease, substantiating current clinical trials of therapeutic interventions targeting alterations in brain levels of these metals in Parkinson's disease.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science.

The recent discovery and subsequent development of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease.

Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

Geometric regulation of histone state directs melanoma reprogramming.

Malignant melanoma displays a high degree of cellular plasticity during disease progression. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation. Here we uncover a relationship between geometric features at perimeter regions of melanoma aggregates, and reprogramming to a stem cell-like state through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find spatial enrichment of these histone modifications with concurrent expression of stemness markers. The epigenetic modifier PRDM14 overlaps with H3K9Ac and shows elevated expression in cells along regions of perimeter curvature. siRNA knockdown of PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma aggregates may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.

Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell-based cartilage tissue engineering.

Despite great progress in biomaterial design strategies for replacing damaged articular cartilage, prevention of stem cell-derived chondrocyte hypertrophy and resulting inferior tissue formation is still a critical challenge. Here, by using engineered biomaterials and a high-throughput system for screening of combinatorial cues in cartilage microenvironments, we demonstrate that biomaterial cross-linking density that regulates matrix degradation and stiffness-together with defined presentation of growth factors, mechanical stimulation, and arginine-glycine-aspartic acid (RGD) peptides-can guide human mesenchymal stem cell (hMSC) differentiation into articular or hypertrophic cartilage phenotypes. Faster-degrading, soft matrices promoted articular cartilage tissue formation of hMSCs by inducing their proliferation and maturation, while slower-degrading, stiff matrices promoted cells to differentiate into hypertrophic chondrocytes through Yes-associated protein (YAP)-dependent mechanotransduction. in vitro and in vivo chondrogenesis studies also suggest that down-regulation of the Wingless and INT-1 (WNT) signaling pathway is required for better quality articular cartilage-like tissue production.

A pH-responsive silica-metal-organic framework hybrid nanoparticle for the delivery of hydrophilic drugs, nucleic acids, and CRISPR-Cas9 genome-editing machineries.

Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica-metal-organic framework hybrid nanoparticle (SMOF NP) consisting of both silica and zeolitic imidazole framework (ZIF). This unique SMOF NP offers a high loading content and efficiency, excellent stability, and robust intracellular delivery of a variety of payloads, including hydrophilic small molecule drugs (e.g., doxorubicin hydrochloride), nucleic acids (e.g., DNA and mRNA), and genome-editing machineries (e.g., Cas9-sgRNA ribonucleoprotein (RNP), and RNP together with donor DNA (e.g., RNP + ssODN)). The superior drug delivery/gene transfection/genome-editing efficiencies of the SMOF NP are attributed to its pH-controlled release and endosomal escape capabilities due to the proton sponge effect enabled by the imidazole moieties in the SMOF NPs. Moreover, the surface of the SMOF NP can be easily customized (e.g., PEGylation and ligand conjugation) via various functional groups incorporated into the silica component. RNP-loaded SMOF NPs induced efficient genome editing in vivo in murine retinal pigment epithelium (RPE) tissue via subretinal injection, providing a highly promising nanoplatform for the delivery of a wide range of hydrophilic payloads.

A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing.

Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM).

The development of three-dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label-free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion-relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass-transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.

Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery.

Gene therapy holds great promise for the treatment of many diseases, but clinical translation of gene therapies has been slowed down by the lack of safe and efficient gene delivery systems. Here, we report two versatile redox-responsive polyplexes (i.e., cross-linked and non-crosslinked) capable of efficiently delivering a variety of negatively charged payloads including plasmid DNA (DNA), messenger RNA, Cas9/sgRNA ribonucleoprotein (RNP), and RNP-donor DNA complexes (S1mplex) without any detectable cytotoxicity. The key component of both types of polyplexes is a cationic poly( N, N'-bis(acryloyl)cystamine- co-triethylenetetramine) polymer [a type of poly( N, N'-bis(acryloyl)cystamine-poly(aminoalkyl)) (PBAP) polymer] containing disulfide bonds in the backbone and bearing imidazole groups. This composition enables efficient encapsulation, cellular uptake, controlled endo/lysosomal escape, and cytosolic unpacking of negatively charged payloads. To further enhance the stability of non-crosslinked PBAP polyplexes, adamantane (AD) and ß-cyclodextrin (ß-CD) were conjugated to the PBAP-based polymers. The cross-linked PBAP polyplexes formed by host-guest interaction between ß-CD and AD were more stable than non-crosslinked PBAP polyplexes in the presence of polyanionic polymers such as serum albumin, suggesting enhanced stability in physiological conditions. Both cross-linked and non-crosslinked polyplexes demonstrated either similar or better transfection and genome-editing efficiencies, and significantly better biocompatibility than Lipofectamine 2000, a commercially available state-of-the-art transfection agent that exhibits cytotoxicity.

Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.

Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.

Melanoma topology reveals a stem-like phenotype that promotes angiogenesis.

Tumor angiogenesis provides critical nutrients for cancer progression and may also facilitate pathways for dissemination during the process of metastasis. It is well established that cells that metastasize display characteristics of stem cells; however, the prevailing paradigm points to these stem-like cells residing in the hypoxic niche within the tumor interior. Controlling the geometry at the interface of a population of melanoma cells reveals a role for perimeter topology in promoting a stem-like state with enhanced tumorigenicity. We show that this putative melanoma-initiating cell (MIC) demonstrates significant enhancement in the secretion of proangiogenic molecules. This finding suggests the possibility of an "invasive niche" at the perimeter of a growing tumor that promotes a MIC state with angiogenic activity. Using several in vitro and in vivo models of tumor angiogenesis, we see concurrent stem-like characteristics with initiation of neovascularization. In the absence of hypoxia, precise topological cues induce signaling through integrin α5ß1 and downstream extracellular signal-regulated kinase (ERK) signaling to regulate the MIC secretome through the signal transducer and activator of transcription (STAT) and hypoxia-inducible factor 1α (HIF1α) pathways. Inhibiting integrin α5ß1 and ERK signaling attenuates both the MIC phenotype and proangiogenic signaling. These results suggest that topological cues in the periphery of malignant melanoma promote the MIC state-using mechanotransduction in lieu of low oxygen-to facilitate the formation of new vasculature for progression and invasion.

Manufacturing Cell Therapies Using Engineered Biomaterials.

Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tunable properties offer an intriguing tool to provide or deliver cues to retain stemness, direct differentiation, promote reprogramming, manipulate the genome, or select functional phenotypes. This review discusses the use of engineered biomaterials to control human cell manufacturing. Future work exploiting engineered biomaterials has the potential to generate manufacturing processes that produce standardized cells with well-defined critical quality attributes appropriate for clinical testing.

Anti-inflammatory ω-3 endocannabinoid epoxides.

Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

UNLABELLED: Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvß3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes.

Temporal Modulation of Stem Cell Activity Using Magnetoactive Hydrogels.

Cell activity is coordinated by dynamic interactions with the extracellular matrix, often through stimuli-mediated spatiotemporal stiffening and softening. Dynamic changes in mechanics occur in vivo through enzymatic or chemical means, processes which are challenging to reconstruct in cell culture materials. Here a magnetoactive hydrogel material formed by embedding magnetic particles in a hydrogel matrix is presented whereby elasticity can be modulated reversibly by attenuation of a magnetic field. Orders of magnitude change in elasticity using low magnetic fields are shown and reversibility of stiffening with simple permanent magnets is demonstrated. The broad applicability of this technique is demonstrated with two therapeutically relevant bioactivities in mesenchymal stem cells: secretion of proangiogenic molecules, and dynamic control of osteogenesis. The ability to reversibly stiffen cell culture materials across the full spectrum of soft tissue mechanics, using simple materials and commercially available permanent magnets, makes this approach viable for a broad range of laboratory environments.

Interfacial geometry dictates cancer cell tumorigenicity.

Within the heterogeneous architecture of tumour tissue there exists an elusive population of stem-like cells that are implicated in both recurrence and metastasis. Here, by using engineered extracellular matrices, we show that geometric features at the perimeter of tumour tissue will prime a population of cells with a stem-cell-like phenotype. These cells show characteristics of cancer stem cells in vitro, as well as enhanced tumorigenicity in murine models of primary tumour growth and pulmonary metastases. We also show that interfacial geometry modulates cell shape, adhesion through integrin α5ß1, MAPK and STAT activity, and initiation of pluripotency signalling. Our results for several human cancer cell lines suggest that interfacial geometry triggers a general mechanism for the regulation of cancer-cell state. Similar to how a growing tumour can co-opt normal soluble signalling pathways, our findings demonstrate how cancer can also exploit geometry to orchestrate oncogenesis.

Capturing extracellular matrix properties in vitro: Microengineering materials to decipher cell and tissue level processes.

Rapid advances in biology have led to the establishment of new fields with tremendous translational potential including regenerative medicine and immunoengineering. One commonality to these fields is the need to extract cells for manipulation in vitro; however, results obtained in laboratory cell culture will often differ widely from observations made in vivo. To more closely emulate native cell biology in the laboratory, designer engineered environments have proved a successful methodology to decipher the properties of the extracellular matrix that govern cellular decision making. Here, we present an overview of matrix properties that affect cell behavior, strategies for recapitulating important parameters in vitro, and examples of how these properties can affect cell and tissue level processes, with emphasis on leveraging these tools for immunoengineering.