Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection.

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.

Naturally occurring T cell mutations enhance engineered T cell therapies.

Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.

Functional improvements of patients admitted to an inpatient rehabilitation facility after bilateral lung transplant due to severe COVID-19 pulmonary disease.

BACKGROUND: Coronavirus disease (COVID-19) has introduced a new subset of patients with acute end-stage lung damage for which lung transplantation has been successfully performed. OBJECTIVE: To describe the inpatient rehabilitation course of patients who underwent bilateral lung transplant due to severe COVID-19 pulmonary disease. DESIGN: Retrospective chart review. SETTING: Free-standing, academic, urban inpatient rehabilitation hospital. PARTICIPANTS: Seventeen patients aged 28-67 years old (mean 53.9 ± 10.7) who developed COVID-19 respiratory failure and underwent bilateral lung transplant. INTERVENTIONS: Patients participated in a comprehensive inpatient rehabilitation program including physical, occupational, and speech therapy tailored to the unique functional needs of each individual. MAIN OUTCOME MEASURES: Primary outcome measures of functional improvements, include mobility and self-care scores on section GG of the Functional Abilities and Goals of the Improving Post-Acute Care Transformation Act, as defined as quality measures by the Centers for Medicare and Medicaid Services. Other functional measures included 6 minute walk test, Berg balance scale, Mann Assessment of Swallowing Ability (MASA), and Cognition and Memory Functional Independence Measure (FIM) scores. Wilcoxon signed rank sum test was used to evaluate statistical significance of change between admission and discharge scores. RESULTS: Fourteen patients completed inpatient rehabilitation. Self-care (GG0130) mean score improved from 20.9 to 36.1. Mobility (GG0170) mean score improved from 30.7 to 70.7. Mean 6-minute walk distance improved from 174.1 to 467.6 feet. Mean Berg balance scores improved from 18.6/56 to 36.3/56. MASA scores improved from 171.3 to 182.3. All functional measures demonstrated statistically significant improvements with p value ≤ .008, except for cognition and memory FIM scores, which did not show a statistically significant difference. A majority (76%) of patients discharged home. CONCLUSION: This new and unique patient population can successfully participate in a comprehensive inpatient rehabilitation program and achieve functional improvements despite medical complications.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

The Relationship Between Patient-Specific Factors and Discharge Destination After COVID-19 Hospitalization.

OBJECTIVE: The aim of this study was to determine the discharge destinations and associated patient-specific factors among patients hospitalized with COVID-19. DESIGN: A retrospective cohort study was carried out at a single-site tertiary acute care hospital. RESULTS: Among 2872 patients, discharge destination included home without services ( n = 2044, 71.2%), home with services ( n = 379, 13.2%), skilled nursing facility (117, 4.1%), long-term acute care hospital ( n = 39, 1.3%), inpatient rehabilitation facility ( n = 97, 3.4%), acute care facility ( n = 23, 0.8%), hospice services ( n = 20, 0.7%), or deceased during hospitalization ( n = 153, 5.3%). Adjusting by covariates, patients had higher odds of discharge to a rehabilitation facility (skilled nursing facility, long-term acute care hospital, or inpatient rehabilitation facility) than home (with or without services) when they were older (odds ratio [OR], 2.37; 95% confidence interval [CI], 1.80-3.11; P < 0.001), had a higher Charlson Comorbidity Index score (3-6: OR, 2.36; 95% CI, 1.34-4.15; P = 0.003; ≥7: OR, 2.76; 95% CI, 1.56-4.86; P < 0.001), were intubated or required critical care (OR, 2.15; 95% CI, 1.48-3.13; P < 0.001), or had a longer hospitalization (3-7 days: OR, 12.48; 95% CI, 3.77-41.32; P < 0.001; 7-14 days: OR, 28.14; 95% CI, 8.57-92.43; P < 0.001). Patients were less likely to be discharged to a rehabilitation facility if they received remdesivir (OR, 0.44; 95% CI, 0.31-0.64; P < 0.001). CONCLUSIONS: Patient-specific factors associated with COVID-19 hospitalization should be considered by physicians when prognosticating patient rehabilitation.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Left ventricular assist devices: A historical perspective at the intersection of medicine and engineering.

Over the last half-century, left ventricular assist device (LVAD) technology has progressed from conceptual therapy for failed cardiopulmonary bypass weaning to an accepted destination therapy for advanced heart failure. The history of LVAD engineering is defined by an initial development phase, which demonstrated the feasibility of such an approach, to the more recent three major generations of commercial devices. In this review, we explore the engineering challenges of LVADs, how they were addressed over time, and the clinical outcomes that resulted from each major technological development. The first generation of commercial LVADs were pulsatile devices, which lacked the appropriate durability due to their number of moving components and hemocompatibility. The second generation of LVADs was defined by replacement of complex, pulsatile pumps with primarily axial, continuous-flow systems with an impeller in the blood passageway. These devices experienced significant commercial success, but the presence of excessive trauma to the blood and in-situ bearing resulted in an unacceptable burden of adverse events. Third generation centrifugal-flow pumps use magnetically suspended rotors within the pump chamber. Superior outcomes with this newest generation of devices have been observed, particularly with respect to hemocompatibility-related adverse events including pump thrombosis, with fully magnetically levitated devices. The future of LVAD engineering includes wireless charging foregoing percutaneous drivelines and more advanced pump control mechanisms, including synchronization of the pump flow with the native cardiac cycle, and varying pump output based on degree of physical exertion using sensor or advanced device-level data triggers.

De Ritis-adjusted AST provides comparable efficacy to lactate dehydrogenase as a biomarker for detection of LVAD hemolysis or thrombosis.

BACKGROUND: Lactate hydrogenase (LDH) is a common biomarker utilized in the detection and monitoring of left ventricular assist device (LVAD) hemolysis and thrombosis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are common laboratory tests that can be used to calculate the De Ritis ratio and the De Ritis adjusted AST. METHODS: A retrospective review of LVAD patients was performed to identify three cohorts of patients: those with confirmed pump thrombosis after device exchange, those with LVAD-related hemolysis who were medically managed without pump exchange, and those who did not meet these criteria and served as the control cohort. Evaluation of AST, AST/ALT ratio (referred to as the De Ritis ratio) as well as AST x (AST/ALT) or the De Ritis-adjusted AST (DRA) was performed. RESULTS: There were 29 patients who underwent device exchange for thrombosis, 25 patients who were diagnosed with hemolysis and treated medically (clopidogrel (N = 6), heparin (N = 13), tirofiban (N = 8), eptifibatide (N = 2), and some received more than one of these treatments), and 425 control patients. A qualitatively comparable relative and absolute rise in DRA and LDH were found in both surgically managed pump thrombosis and suspected device-related hemolysis. CONCLUSIONS: Both AST and LDH as well DRA are significantly associated with pump thrombosis (p < 0.0001 for each). DRA is a potential screening biomarker for hemolysis and device thrombosis in stable left ventricular assist device patients.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells.

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

Glomerular filtration and podocyte tensional homeostasis: importance of the minor type IV collagen network.

The minor type IV collagen chain, which is a significant component of the glomerular basement membrane in healthy individuals, is known to assemble into large structures (supercoils) that may contribute to the mechanical stability of the collagen network and the glomerular basement membrane as a whole. The absence of the minor chain, as in Alport syndrome, leads to glomerular capillary demise and eventually to kidney failure. An important consideration in this problem is that the glomerular capillary wall must be strong enough to withstand the filtration pressure and porous enough to permit filtration at reasonable pressures. In this work, we propose a coupled feedback loop driven by filtration demand and tensional homeostasis of the podocytes forming the outer portion of the glomerular capillary wall. Briefly, the deposition of new collagen increases the stiffness of basement membrane, helping to stress shield the podocytes, but the new collagen also decreases the permeability of the basement membrane, requiring an increase in capillary transmural pressure drop to maintain filtration; the resulting increased pressure outweighs the increased glomerular basement membrane stiffness and puts a net greater stress demand on the podocytes. This idea is explored by developing a multiscale simulation of the capillary wall, in which a macroscopic (µm scale) continuum model is connected to a set of microscopic (nm scale) fiber network models representing the collagen network and the podocyte cytoskeleton. The model considers two cases: healthy remodeling, in which the presence of the minor chain allows the collagen volume fraction to be increased by thickening fibers, and Alport syndrome remodeling, in which the absence of the minor chain allows collagen volume fraction to be increased only by adding new fibers to the network. The permeability of the network is calculated based on previous models of flow through a fiber network, and it is updated for different fiber radii and volume fractions. The analysis shows that the minor chain allows a homeostatic balance to be achieved in terms of both filtration and cell tension. Absent the minor chain, there is a fundamental change in the relation between the two effects, and the system becomes unstable. This result suggests that mechanobiological or mechanoregulatory therapies may be possible for Alport syndrome and other minor chain collagen diseases of the kidney.

Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency.

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).

Lack of Effect of Rezafungin on QT/QTc Interval in Healthy Subjects.

Rezafungin is a new echinocandin in development for treatment of candidemia and invasive candidiasis, and for prophylaxis of invasive fungal infections. Rezafungin is the first echinocandin to undergo definitive QT/QTc study. This phase 1, single-center, randomized, double-blind trial was conducted to assess effects of intravenous rezafungin vs intravenous placebo (with moxifloxacin as positive control) on the QT interval of the electrocardiogram, corrected for heart rate by Fridericia's formula (QTcF), in healthy adults. Therapeutic (600 mg) and supratherapeutic (1400 mg) rezafungin doses were selected to achieve exposures 2.5-fold higher than produced by the highest dose used in a phase 2 trial (400 mg once weekly). The primary end point was change in QTcF from baseline (ΔQTcF) as a function of plasma concentration, assessed by comparing upper bounds of the 2-sided 90% confidence interval. The estimated mean ΔΔQTcF at the mean plasma concentrations for the rezafungin doses had upper bounds <10 milliseconds, within the upper bound of the 2-sided 90% confidence interval. Intravenous rezafungin up to 1400 mg in a single dose did not prolong QT interval and had no apparent effect on repolarization or QRS duration. Electrocardiogram results showed no clinically significant effects of concern. These findings support the continued development of rezafungin.

Outcomes of Acute Inpatient Rehabilitation of Patients With Left Ventricular Assist Devices and Subsequent Stroke.

OBJECTIVE: The aim of the study was to examine the functional outcomes and medical complications of patients with left ventricular assist device implantation and subsequent stroke during comprehensive inpatient rehabilitation. DESIGN: Retrospective cohort study of 21 patients admitted to an inpatient rehabilitation facility between 2009 and 2015. Main outcome measurements include admission and discharge Functional Independence Measure, length of stay, and Functional Independence Measure efficiency. RESULTS: The study included 17 male and 4 female patients aged 32-75 yrs. Eleven patients (52%) required transfer to an acute care hospital for evaluation. Fifteen patients completed inpatient rehabilitation with median [interquartile range] length of stay 26 [13.5-34] days (range = 7-59 days), median [interquartile range] Functional Independence Measure gain of 18 [12.5-32], and median [interquartile range] Functional Independence Measure efficiency of 1.0 [0.6-1.44]. Patients who required transfer to acute care during their course but ultimately completed inpatient rehabilitation (n = 5) demonstrated larger median [interquartile range] Functional Independence Measure gain (40 [23-42]) and longer median [interquartile range] length of stay (35 [35-42]) compared with patients who completed inpatient rehabilitation without transfer (Functional Independence Measure gain = 15 [9.25-26.5]; length of stay = 14.5 [11.5-26.25]). CONCLUSIONS: Patients with left ventricular assist device implantation and subsequent stroke demonstrate functional gains during acute inpatient rehabilitation programs. A large percent of patients required transfer to acute care.