An "off-the-shelf" CD2 universal CAR-T therapy for T-cell malignancies.

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic "universal" CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo.

Pharmacological interventions enhance virus-free generation of -replaced CAR T cells.

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant () locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts.

Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair.

CRISPR-Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell ( ) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications.

CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing.

CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair fingerprints that result from non-homologous end joining (NHEJ) after double-stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis.

Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology.

Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System.

Genome editing of human cluster of differentiation 34 (CD34) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, genome editing using the CRISPR-Cas9 system in CD34 HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34 HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes.

Rational Design of Small Molecules to Enhance Genome Editing Efficiency by Selectively Targeting Distinct Functional States of CRISPR-Cas12a.

CRISPR-Cas12a, a type-V CRISPR-Cas endonuclease, is an effective genome editing platform. To improve the gene editing efficiency of Cas12a, we rationally designed small molecule enhancers through a combined computational approach. First, we used extensive molecular dynamics (MD) simulations to explore the conformational landscape of Cas12a from (AsCas12a), revealing distinct conformational states that could be targeted by small molecules to modulate its genome editing function.

A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method.

Dystroglycan Maintains Inner Limiting Membrane Integrity to Coordinate Retinal Development.

Proper neural circuit formation requires the precise regulation of neuronal migration, axon guidance, and dendritic arborization. Mutations affecting the function of the transmembrane glycoprotein dystroglycan cause a form of congenital muscular dystrophy that is frequently associated with neurodevelopmental abnormalities. Despite its importance in brain development, the role of dystroglycan in regulating retinal development remains poorly understood.

Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes.

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects.

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy.

Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy.

Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry.

Distinct functions of glial and neuronal dystroglycan in the developing and adult mouse brain.

Cobblestone (type II) lissencephaly and mental retardation are characteristic features of a subset of congenital muscular dystrophies that include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama-type congenital muscular dystrophy. Although the majority of clinical cases are genetically undefined, several causative genes have been identified that encode known or putative glycosyltransferases in the biosynthetic pathway of dystroglycan. Here we test the effects of brain-specific deletion of dystroglycan, and show distinct functions for neuronal and glial dystroglycan.

Visual impairment in the absence of dystroglycan.

Ocular involvement in muscular dystrophy ranges from structural defects to abnormal electroretinograms. While the mechanisms underlying the abnormal retinal physiology in patients are not understood, it is thought that alpha-dystroglycan extracellular interactions are critical for normal visual function. Here we show that beta-dystroglycan anchors dystrophin and the inward rectifying K(+) channel Kir4.

Generation and characterization of transgenic mice with the full-length human DMD gene.

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice.

Gene expression profiling highlights defective myogenesis in DMD patients and a possible role for bone morphogenetic protein 4.

Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle weakness and wasting. Despite the sustained presence of satellite cells in their skeletal muscles, muscle regeneration in DMD patients seems inefficient and unable to compensate for the continuous muscle fiber loss. To find a molecular explanation, we compared the gene expression profiles of myoblasts from healthy individuals and DMD patients during activation and differentiation in culture.

Gene expression profiling to monitor therapeutic and adverse effects of antisense therapies for Duchenne muscular dystrophy.

Objectives: The objective of this study was to assess the utility of the gene expression profiling technique for the preclinical evaluation of drug efficacy and safety, taking a new therapeutic approach for Duchenne muscular dystrophy (DMD) as an example.

Methods: Muscles from dystrophin-deficient (mdx) mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the Dmd gene. Synthetic antisense oligonucleotides (AONs) complexed with different carriers to enhance cellular uptake and recombinant adeno-associated virus (rAAV)-expressed antisense sequences were evaluated.

Exploiting the full power of temporal gene expression profiling through a new statistical test: application to the analysis of muscular dystrophy data.

Background: The identification of biologically interesting genes in a temporal expression profiling dataset is challenging and complicated by high levels of experimental noise. Most statistical methods used in the literature do not fully exploit the temporal ordering in the dataset and are not suited to the case where temporal profiles are measured for a number of different biological conditions. We present a statistical test that makes explicit use of the temporal order in the data by fitting polynomial functions to the temporal profile of each gene and for each biological condition.

Large-scale gene expression analysis of human skeletal myoblast differentiation.

To study pathways involved in human skeletal myogenesis, we profiled gene expression in human primary myoblast cells derived from three individuals using both oligonucleotide and cDNA microarrays. Following stringent statistical testing (false-positive rate 0.4%), we identified 146 genes differentially expressed over time.

Gene expression variation between mouse inbred strains.

Background: In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays.

Results: Through ANOVA analysis with a false discovery rate of 10%, we show that 1.