A novel gene editing system to treat both Tay-Sachs and Sandhoff diseases.

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme ß-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex ß subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p < 0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p < 0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay-Sachs and Sandhoff patients.

Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.

Deficiencies in the lysosomal hydrolase ß-galactosidase (ß-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse ß-gal encoding gene, Glb1, was targeted to generate both models of ß-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of ß-gal (ß-gal-/-). Glb1W274L mice showed a significant reduction in ß-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, ß-gal-/- mice were devoid of ß-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). ß-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of ß-galactosidase deficiency with residual enzyme activity has been developed.

Examination of a blood-brain barrier targeting ß-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase ß-galactosidase (ß-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing ß-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (ß-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult ß-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in ß-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating ß-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated ß-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed ß-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in ß-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active ß-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to ß-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in ß-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets.

In the present study, phospholipase A(2) (PLA(2))-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA(2) activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanied by increased arachidonic acid and thromboxane B(2) release and increased lysoplasmenylcholine production. Thrombin stimulation caused an increase in PLA(2) activity measured in the cytosolic fraction with plasmenylcholine only; no increase in activity was measured with phosphatidylcholine. No change in membrane-associated PLA(2) activity was observed with either substrate tested. Pretreatment with the Ca(2+)-independent PLA(2)-selective inhibitor, bromoenol lactone, inhibited completely any thrombin-stimulated phospholipid hydrolysis. Thus, thrombin stimulation of human platelets activates a cytosolic PLA(2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids.

Protease-activated receptor stimulation activates a Ca2+-independent phospholipase A2 in bladder microvascular endothelial cells.

Increased mast cell numbers and mast cell activation represent one of the prevalent etiologic theories for interstitial cystitis, an inflammatory condition in the bladder. This study was designed primarily to determine whether increased mast cell tryptase in the bladder wall may play a role in activating bladder endothelial cell phospholipase A(2) (PLA(2)), leading to increased inflammatory phospholipid metabolite accumulation, which may propagate the inflammatory process. We stimulated human bladder microvascular endothelial cells with thrombin or tryptase and measured the activation of PLA(2) and the production of multiple membrane phospholipid-derived inflammatory mediators. Thrombin and tryptase stimulation resulted in activation of a Ca(2+)-independent PLA(2), leading to increased release of arachidonic acid and prostacyclin and increased production of platelet-activating factor. These responses were blocked completely by pretreatment of human bladder microvascular endothelial cells with the Ca(2+)-independent PLA(2)-selective inhibitor bromoenol lactone. The combination of increased prostacyclin and platelet-activating factor in the bladder circulation may result in vasodilation and increased polymorphonuclear leukocyte adherence to the endothelium and may facilitate recruitment of polymorphonuclear leukocytes to the bladder wall of patients with interstitial cystitis.

Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells.

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 +/- 0.1 vs. 5.3 +/- 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 +/- 1.0 vs. 12.0 +/- 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 +/- 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 +/- 1.0 nmol PO4/mg of protein). Incubation of HCAEC with the selective iPLA2 inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 +/- 0.3 vs. 1.1 +/- 0.1%) and increased production of lysoplasmenylcholine (1.4 +/- 0.2 vs. 0.6 +/- 0.1 nmol PO4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 +/- 0.6 to 0.5 +/- 0.1%) and lysoplasmenylcholine production (2.0 +/- 0.1 to 0.2 +/- 0.1 nmol PO4/mg of protein). These data suggest that PKC activates iPLA2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production.

Inhibition of platelet-activating factor (PAF) acetylhydrolase by methyl arachidonyl fluorophosphonate potentiates PAF synthesis in thrombin-stimulated human coronary artery endothelial cells.

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.