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1.
Sci Rep ; 8(1): 16304, 2018 11 02.
Article in English | MEDLINE | ID: mdl-30389991

ABSTRACT

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.


Subject(s)
Fetal Hemoglobin/genetics , Filtration/methods , Gene Editing/methods , Hematopoietic Stem Cell Transplantation , Ribonucleoproteins/genetics , Animals , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Cells, Cultured , Electroporation/methods , Female , Fetal Hemoglobin/metabolism , Filtration/economics , Filtration/instrumentation , Genetic Therapy/economics , Genetic Therapy/instrumentation , Genetic Therapy/methods , Hematopoietic Stem Cells/metabolism , Hemoglobinopathies/genetics , Hemoglobinopathies/therapy , Humans , Membranes, Artificial , Mice , Models, Animal , RNA, Guide, Kinetoplastida/genetics , Transplantation, Autologous
2.
Tomography ; 4(2): 43-45, 2018 Jun.
Article in English | MEDLINE | ID: mdl-30206543

ABSTRACT

Prenatal ultrasonography in the early third trimester showed an unusual branching pattern of the right aortic arch. Echocardiography performed 4 h after birth showed the right aortic arch with mirror-image branching, patent ductus arteriosus, and patent foramen ovale. Because the location of the ductus arteriosus was unclear on echocardiography, cardiovascular magnetic resonance imaging was performed 3 days after birth. Advanced techniques including contrast-enhanced time-resolved magnetic resonance angiography and 3D time-of-flight magnetic resonance angiography allowed accurate diagnosis of a vascular ring comprising ascending and descending aorta, right aortic arch with mirror-image branching, and diverticulum of Kommerell giving rise to a left ligamentum arteriosum. The infant had hiccups, but no other symptoms. The esophagram was negative for obstruction. The infant was closely monitored; however, she developed esophageal obstruction at 7 months of age because of the vascular ring. She underwent lysis of the left ligamentum arteriosum followed by aortopexy for relief of esophageal obstruction. This report shows the utility of neonatal cardiovascular magnetic resonance imaging to evaluate complex congenital aortic arch anomalies.

3.
ACS Med Chem Lett ; 3(1): 74-8, 2012 Jan 12.
Article in English | MEDLINE | ID: mdl-24900374

ABSTRACT

The optimization of a series of S1P1 agonists with limited activity against S1P3 is reported. A polar headgroup was used to improve the physicochemical and pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood lymphocytes 24 h postdose. In rats, a dose-proportional increase in exposure was measured when 22 was dosed orally at 2 and 100 mg/kg.

4.
Bioorg Med Chem Lett ; 22(1): 527-31, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22104144

ABSTRACT

We reveal how a N-scan SAR strategy (systematic substitution of each CH group with a N atom) was employed for quinolinone-based S1P(1) agonist 5 to modulate physicochemical properties and optimize in vitro and in vivo activity. The diaza-analog 17 displays improved potency (hS1P(1) RI; 17: EC(50)=0.020 µM, 120% efficacy; 5: EC(50)=0.070 µM, 110% efficacy) and selectivity (hS1P(3) Ca(2+) flux; 17: EC(50) >25 µM; 5: EC(50)=1.5 µM, 92% efficacy), as well as enhanced pharmacokinetics (17: CL=0.15 L/h/kg, V(dss)=5.1L/kg, T(1/2)=24h, %F=110; 5: CL=0.93L/h/kg, V(dss)=11L/kg, T(1/2)=15 h, %F=60) and pharmacodynamics (17: 1.0mg/kg po, 24h PLC POC=-67%; 5: 3mg/kg po, 24h PLC POC=-51%) in rat.


Subject(s)
Chemistry, Physical/methods , Quinolones/pharmacology , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/chemistry , Animals , Area Under Curve , Cardiovascular Diseases/metabolism , Drug Design , Female , Humans , Immunosuppressive Agents/pharmacology , In Vitro Techniques , Kinetics , Lymphocytes/cytology , Lymphocytes/metabolism , Models, Chemical , Multiple Sclerosis/drug therapy , Quinolones/chemistry , Rats , Rats, Inbred Lew , Structure-Activity Relationship
5.
ACS Med Chem Lett ; 2(10): 752-7, 2011 Oct 13.
Article in English | MEDLINE | ID: mdl-24900263

ABSTRACT

The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist lacking a polar headgroup, which is shown to effect dramatic reduction of circulating lymphocytes (POC = -78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent potency that 8 exhibits toward S1P1 (EC50 = 0.035 µM, 96% efficacy) and the >100-fold selectivity that it displays against receptor subtypes S1P2-5 suggest that it may serve as a valuable tool to understand the clinical relevance of selective S1P1 agonism.

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