Browsing by Author "Yao, Jing"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Characterization, Enrichment, and Computational Modeling of Cross-Linked Actin Networks in Transformed Trabecular Meshwork Cells
    (Association for Research in Vision and Ophthalmology, 2025) Li, Haiyan; Harvey, Devon H.; Dai, Jiannong; Swingle, Steven P.; Compton, Anthony M.; Sugali, Chenna Kesavulu; Dhamodaran, Kamesh; Yao, Jing; Lin, Tsai-Yu; Sulchek, Todd; Kim, Taeyoon; Ethier, C. Ross; Mao, Weiming; Ophthalmology, School of Medicine
    Purpose: Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM). We previously developed the GTM3L cell line, which spontaneously forms fluorescently labeled CLANs, by transducing GTM3, a transformed glaucomatous TM cell line, with a lentivirus expressing the LifeAct-GFP fusion protein. Here, we determined if LifeAct-GFP viral copy numbers are associated with CLANs, developed approaches to increase CLAN incidence, and computationally studied the biomechanical properties of CLAN-containing GTM3L cells. Methods: GTM3L cells were fluorescently sorted for viral copy number analysis to determine whether increased CLAN incidence was associated with copy number. CLAN incidence was increased by combining (1) differential adhesion sorting, (2) cell deswelling, and (3) cell stiffness selection. GTM3L cells were cultured on glass or soft hydrogels for stiffness measurement by atomic force microscopy. Computational models studied the biomechanical properties of CLANs. Results: GTM3L cells had one LifeAct-GFP viral copy/cell on average, and viral copy number or LifeAct-GFP expression level did not associate with CLAN incidence rate. However, CLAN rate was increased from -0.28% to -50% by combining the three enrichment methods noted above. Further, GTM3L cells formed more CLANs on a stiff versus a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. Conclusions: It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the biomechanics, mechanobiology, and etiology of CLANs in the TM.
  • Thumbnail Image
    Item
    Real-Time PCR: An Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells
    (Elsevier, 2004-12-04) Villella, Anthony D.; Yao, Jing; Getty, Robert R.; Juliar, Beth E.; Yiannoutsos, Constantin; Hartwell, Jennifer R.; Cai, Shanbao; Sadat, Mohammed A.; Cornetta, Kenneth; Williams, David A.; Pollok, Karen E.; Medical and Molecular Genetics, School of Medicine
    Accurate measurement of gene transfer into hematopoietic progenitor cells is an essential prerequisite for assessing the utility of gene therapy approaches designed to correct hematologic defects. We developed a reliable method to measure transduction efficiency at the level of the progenitor cell with real-time polymerase chain reaction (PCR) analysis of individual progenitor-derived colonies. We hypothesized that this method would demonstrate better sensitivity and specificity than are currently achievable with conventional PCR. An oncoretroviral vector containing the enhanced green fluorescent protein was used to transduce human CD34+ cells derived from bone marrow or granulocyte-colony-stimulating factor-mobilized peripheral blood. Progenitor assays were set up and colonies plucked after visualization by fluorescence microscopy. By analyzing microscopically identified fluorescent samples and nontransduced samples, we calculated an overall sensitivity and specificity of 90.2 and 95.0%, respectively. Real-time PCR had higher specificity and sensitivity than conventional PCR as analyzed by generalized linear models (P = 0.002 and P = 0.019, respectively). In conclusion, we found real-time PCR to have superior sensitivity and specificity compared to conventional PCR in determining transduction efficiency of hematopoietic progenitor cells.
  • Thumbnail Image
    Item
    Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials
    (Elsevier, 2023) Cornetta, Kenneth; Yao, Jing; House, Kimberley; Duffy, Lisa; Adusumilli, Prasad S.; Beyer, Rachel; Booth, Claire; Brenner, Malcolm; Curran, Kevin; Grilley, Bambi; Heslop, Helen; Hinrichs, Christian S.; Kaplan, Rosandra N.; Kiem, Hans-Peter; Kochenderfer, James; Kohn, Donald B.; Mailankody, Sham; Norberg, Scott M.; O’Cearbhaill, Roisin E.; Pappas, Jennifer; Park, Jae; Ramos, Carlos; Ribas, Antonio; Rivière, Isabelle; Rosenberg, Steven A.; Sauter, Craig; Shah, Nirali N.; Slovin, Susan F.; Thrasher, Adrian; Williams, David A.; Lin, Tsai-Yu; Medical and Molecular Genetics, School of Medicine
    The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.