CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

Non-invasive evaluation of the GABAergic/glutamatergic system in autistic patients observed by MEGA-editing proton MR spectroscopy using a clinical 3 tesla instrument.

Amino acids related to neurotransmitters and the GABAergic/glutamatergic system were measured using a 3 T-MRI instrument in 12 patients with autism and 10 normal controls. All measurements were performed in the frontal lobe (FL) and lenticular nuclei (LN) using a conventional sequence for n-acetyl aspartate (NAA) and glutamate (Glu), and the MEGA-editing method for GABA. The GABA level and [GABA]/[NAA] ratio were significantly lower (p < 0.01) in the FL, but not the LN, in patients with autism compared to normal controls. The [GABA]/[Glu] ratio in the FL was also significantly lower (p < 0.05) in the patients than in the normal controls, thus suggesting a possible abnormality in the regulation between GABA and Glu.

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome.

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA-integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.

Analysis of changes in DNA copy number in radiation-induced thymic lymphomas of susceptible C57BL/6, resistant C3H and hybrid F1 Mice.

Radiation-induced thymic lymphoma in mice is a useful model for studying both the mechanism of radiation carcinogenesis and genetic susceptibility to tumor development. Using array-comparative genomic hybridization, we analyzed genome-wide changes in DNA copy numbers in radiation-induced thymic lymphomas that had developed in susceptible C57BL/6 and resistant C3H mice and their hybrids, C3B6F1 and B6C3F1 mice. Besides aberrations at known relevant genetic loci including Ikaros and Bcl11b and trisomy of chromosome 15, we identified strain-associated genomic imbalances on chromosomes 5, 10 and 16 and strain-unassociated trisomy of chromosome 14 as frequent aberrations. In addition, biallelic rearrangements at Tcrb were detected more frequently in tumors from C57BL/6 mice than in those from C3H mice, suggesting aberrant V(D)J recombination and a possible link with tumor susceptibility. The frequency and spectrum of these copy-number changes in lymphomas from C3B6F1 and B6C3F1 mice were similar to those in C57BL/6 mice. Furthermore, the loss of heterozygosity analyses of tumors in F(1) mice indicated that allelic losses at Ikaros and Bcl11b were caused primarily by multilocus deletions, whereas those at the Cdkn2a/Cdkn2b and Pten loci were due mainly to uniparental disomy. These findings provide important clues to both the mechanisms for accumulation of aberrations during radiation-induced lymphomagenesis and the different susceptibilities of C57BL/6 and C3H mice.

NAALADase inhibition protects motor neurons against chronic glutamate toxicity.

Glutamate toxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis. The neuropeptide N-acetyl-aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. N-acetylated-alpha-linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II) yields N-acetyl aspartate (NAA) and glutamate. Prior studies have demonstrated NAALADase upregulation in motor cortex and increased NAAG, NAA and glutamate in cerebrospinal fluid from amyotrophic lateral sclerosis patients. The potent NAALADase inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), was tested in an in vitro model of chronic glutamate-mediated motor neuron degeneration. Neuroprotection was determined (1) biochemically, by measuring choline acetyltransferase activity, (2) immunohistochemically, by counting neurofilament-H-positive motor neurons and (3) morphologically, with phase contrast microscopy. 2-PMPA (10 microM) had significant neuroprotective effects on motor neurons as evidenced by increased choline acetyltransferase activity, decreased motor neuron loss and improved gross morphology. Results suggest that NAALADase inhibitors protect against chronic glutamate-mediated motor neuron degeneration and may prove therapeutic towards amyotrophic lateral sclerosis.

Exocytotic "constipation" is a mechanism of tubulin/lysosomal interaction in colchicine myopathy.

Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis. Thus, we examined, as a possible cellular pathogenesis of colchicine myopathy, how the muscle cytoskeleton affects the degradation of membrane proteins, which are processed through the endosomal/lysosomal pathway. We used the acetylcholine receptor as a model membrane component in cultured myotubes allowed to preincubate with colchicine. We tested at which step colchicine interferes with receptor trafficking by accounting for internalization, delivery to lysosomes, hydrolysis, or exocytotic release of debris. We report that colchicine significantly decreases the exocytosis of AChRs but does not affect receptor internalization, lysosomal hydrolysis, or the number of surface membrane receptors. Further, our immunofluorescence observations revealed a morphologic tubulin network in rat skeletal muscle that is more densely distributed in white (mitochondria-poor) muscle fibers than in red (mitochondria-rich) fibers but is present in both. Ultrastructurally, immunogold labeling localized tubulin in the intermyofibrillar region in a long and linear fashion, unassociated with myofibers or mitochondria. Taken together, our findings suggest the following: (1) Microtubules likely play a functional role in the pathway of lysosomal degradation in normal adult skeletal muscle; (2) The observed decrease in overall apparent degradation of membrane receptors by colchicine must be due primarily to inhibition of exocytosis. These data indicate that lysosomal "constipation" underlies colchicine myopathy. (3) An animal model faithful to the human disorder will allow further pathogenetic studies.

Intracellular fibroblast growth factor produces effects different from those of extracellular application on development of avian cochleovestibular ganglion cells in vitro.

In an avian coculture system, the neuronal precursors of the cochleovestibular ganglion typically migrated from the otocyst and differentiated in response to soluble fibroblast growth factor (FGF-2), which had free access to FGF receptors on the cell surface. Free FGF-2 switched cells from a proliferation mode to migration, accompanied by increases in process outgrowth, fasciculation, and polysialic acid expression. Microsphere-bound FGF-2 had some of the same effects, but in addition it increased proliferation and decreased fasciculation and polysialic acid. As shown by immunohistochemistry, FGF-2 that was bound to latex microspheres depleted the FGF surface receptor protein, which localized with the microspheres in the cytoplasm and nucleus. For microsphere-bound FGF-2, the surface receptor-mediated responses to FGF-2 appear to be limited and the door opened to another venue of intracellular events or an intracrine mechanism.

Identification of the neuroprotective molecular region of pigment epithelium-derived factor and its binding sites on motor neurons.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) family, is a survival factor for various types of neurons. We studied the mechanisms by which human PEDF protects motor neurons from degeneration, with the goal of eventually conducting human clinical trials. We first searched for a molecular region of human PEDF essential to motor neuron protection. Using a spinal cord culture model of chronic glutamate toxicity, we show herein that a synthetic 44 mer peptide from an N-terminal region of the human PEDF molecule that lacks the homologous serpin-reactive region contains its full neuroprotective activity. We also investigated the presence and distribution of PEDF receptors in the spinal cord. Using a fluoresceinated PEDF probe, we show that spinal motor neurons contain specific binding sites for PEDF. Kinetics analyses using a radiolabeled PEDF probe demonstrate that purified rat motor neurons contain a single class of saturable and specific binding sites. This study indicates that a small peptide fragment of the human PEDF molecule could be engineered to contain all of its motor neuron protective activity, and that the neuroprotective action is likely to be mediated directly on motor neurons via a single class of PEDF receptors. The data support the pharmacotherapeutic potential of PEDF as a neuroprotectant in human motor neuron degeneration.

Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis.

Pigment epithelium-derived factor (PEDF), a recently defined retinal trophic factor and anti-angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium-derived factor protein was detected by quantitative western blot analysis with a PEDF-specific antiserum. Levels of PEDF in CSF, expressed as a ratio to total CSF protein, were significantly elevated 3.4-fold in 15 patients with ALS compared with neurologic disease controls (p < 0.0003). This increase does not seem likely to reflect up-regulation of PEDF synthesis in muscle in response to denervation, as CSF PEDF was not elevated in severe denervating diseases other than ALS. Nor does the increase represent some non-specific release in neurodegeneration, as CSF PEDF was not elevated in other neurodegenerative diseases. While the mechanism of this presumably reactive increase is not known, the distinctive, surprisingly elevated level of PEDF in the CSF may be an autoprotective reaction in ALS.