RESUMO
Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for ß-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated ß- globin alleles (ßS/ßS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated ßS to the wild-type ßA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous ßS/ßA hiPSCs, which express the wild-type (ßA) transcript to 30-40% level as compared to uncorrected (ßS/ßS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.
Assuntos
Anemia Falciforme/terapia , Diferenciação Celular , Fibrose Cística/terapia , Endonucleases/metabolismo , Doença de Gaucher/terapia , Terapia Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Anemia Falciforme/genética , Sequência de Bases , Células Cultivadas , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Doença de Gaucher/genética , Proteínas de Fluorescência Verde/genética , Hemoglobinas/genética , Humanos , Fator 4 Semelhante a Kruppel , Dados de Sequência Molecular , Mutação/genética , Receptores CCR5/genética , Medicina RegenerativaRESUMO
This is a comparison review of GeriaSims and Care of the Aging Medical Patient (CHAMP) modules addressing issues in palliative and hospice medicine found in the Portal of Geriatric Online Education, a free on-line repository of geriatric educational materials for medical educators. GeriaSims is a self-directed teaching module designed to systematically address many of the important questions involved in caring for individuals with chronic progressive and life-limiting illnesses. It is well suited for physicians, particularly medical residents and fellows in-training, who provide care for medically complicated elderly and terminally ill individuals. The CHAMP module is designed to familiarize physician educators with palliative and hospice medicine basics to teach residents and fellows through didactic slides, although it can probably be adapted for use by residents and fellows if audio commentary accompanies the slides. Both modules address practical approaches to addressing palliative care in patients and their families. They are useful teaching tools that address an important learning need and can be readily used to supplement current residency curriculum in hospice and palliative medicine.
Assuntos
Instrução por Computador , Currículo , Educação de Pós-Graduação em Medicina , Geriatria/educação , Cuidados Paliativos , Cuidados Paliativos na Terminalidade da Vida , Humanos , Internato e Residência , Simulação de PacienteRESUMO
The in vivo rate of proliferation of Mycobacterium tuberculosis, the causative agent of tuberculosis, has been linked to the rate of progression and severity of disease. Here, we report that deletion of the gene MT2175 (Rv2115c), a putative mycobacterial proteasome-associated AAA-ATPase, leads to a reduction in the growth rate of M. tuberculosis in vitro and in vivo. Despite the reduced growth, the mutant persisted, with slow and gradual clearance in mouse lungs. The mutant elicited reduced levels of interferon-gamma production in the lungs and, when used as an immunizing agent, provided significant protection against challenge with a virulent strain of M. tuberculosis. Expression of the genes lat and MT3159 were highly up-regulated in the mutant. Thus, loss of MT2175 slows both in vitro and in vivo growth rates and compromises the lethality of M. tuberculosis in mice but has a minimal impact on the organism's ability to persist in host tissues.