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Awe J.P.,University of California at Los Angeles | Lee P.C.,University of California at Los Angeles | Ramathal C.,Stanford University | Vega-Crespo A.,University of California at Los Angeles | And 8 more authors.
Stem Cell Research and Therapy | Year: 2013

Introduction. The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. Methods. In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. Results: We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. Conclusion: For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics. © 2013 Awe et al.; licensee BioMed Central Ltd.

Marsden M.D.,University of California at Los Angeles | Avancena P.,University of California at Los Angeles | Hubbard T.,University of California at Los Angeles | Zack J.A.,Immunology and Molecular Genetics
Antimicrobial Agents and Chemotherapy | Year: 2011

CD4 + T cells and macrophages are the primary target cells for HIV in vivo, and antiretroviral drugs can vary in their ability to inhibit the infection of these different cell types. Resistance pathways to the HIV integrase inhibitor raltegravir have previously been investigated in T cells. Primary raltegravir resistance mutations, most often at integrase amino acid position 148 or 155, afford some resistance to the drug. The acquisition of pathway-specific secondary mutations then provides higher-level resistance to viruses infecting T cells. We show here that during macrophage infection, the presence of a single primary raltegravir resistance mutation (Q148H, Q148R, N155H, or N155S) is sufficient to provide resistance to raltegravir comparable to that seen in viruses expressing both primary and secondary mutations in costimulated CD4 + T cells. These data implicate macrophages as a potential in vivo reservoir that may facilitate the development of resistance to raltegravir. Notably, the newer integrase inhibitor MK-2048 effectively suppressed the infection of all raltegravir-resistant viruses in both T cells and macrophages, indicating that more recently developed integrase inhibitors are capable of inhibiting infection in both major HIV cellular reservoirs, even in patients harboring raltegravir-resistant viruses. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Wang L.J.,Immunology and Molecular Genetics | Zhou Z.H.,University of California at Los Angeles
American Laboratory | Year: 2014

Cryo-electron microscopy (Cryo-EM) is particularly favorable for large and transiently stable complexes for which conventional determination methods are unsuitable. Biological samples are prepared using standard purification protocols specific to each sample type of interest and stored in physiological buffer. An aliquot of the sample is applied to an EM grid using a pipet, blotted using filter paper to remove excess sample, and plunged into liquid ethane cooled by liquid nitrogen. Electrons emitted from the electron gun are condensed into a parallel, coherent beam that penetrates the sample loaded in the column. The most important resolution-limiting factor in the cryoEM of biological samples is sample damage caused by bombardment of electrons which, if exceeding 20 electrons/Å2, could break the chemical bonds and cause damage to the sample structures. To obtain a 3-D structure from cryoEM projection images, particles are boxed out and their orientations are determined by image processing.

Keseler I.M.,SRI International | Collado-Vides J.,National Autonomous University of Mexico | Santos-Zavaleta A.,National Autonomous University of Mexico | Peralta-Gil M.,National Autonomous University of Mexico | And 18 more authors.
Nucleic Acids Research | Year: 2011

EcoCyc (http://EcoCyc.org) is a comprehensive model organism database for Escherichia coli K-12 MG1655. From the scientific literature, EcoCyc captures the functions of individual E. coli gene products; their regulation at the transcriptional, post-transcriptional and protein level; and their organization into operons, complexes and pathways. EcoCyc users can search and browse the information in multiple ways. Recent improvements to the EcoCyc Web interface include combined gene/ protein pages and a Regulation Summary Diagram displaying a graphical overview of all known regulatory inputs to gene expression and protein activity. The graphical representation of signal transduction pathways has been updated, and the cellular and regulatory overviews were enhanced with new functionality. A specialized undergraduate teaching resource using EcoCyc is being developed. © The Author(s) 2010.

Bennett M.S.,Immunology and Molecular Genetics | Joseph A.,Immunology and Molecular Genetics | Ng H.L.,Immunology and Molecular Genetics | Goldstein H.,Immunology and Molecular Genetics | And 2 more authors.
AIDS | Year: 2010

Objective: T-cell receptor (TCR) gene therapy is an approach being considered for HIV-1, but epitope mutation is a significant barrier. We assessed whether HIV-specific TCR can be modified to have broader coverage of epitope variants by recombining polymorphisms between public clonotype TCR sequences. Design: Public clonotype TCRs recognizing the same epitope often differ by polymorphisms in their third complementarity determining regions (CDR3). We assessed whether novel combinations of such polymorphisms could improve TCR recognition of epitope variation. Methods: A TCR recognizing the HLA A*0201-restricted epitope SLYNTVATL (Gag 77-85, SL9) was engineered to have combinations of four polymorphisms in the CDR3 regions compared to another SL9-specific TCR. These novel TCRs were screened for functional avidities against SL9 epitope variants and abilities to mediate cytotoxic T-lymphocyte suppression of HIV-1 containing the same epitope variants. Results: The TCRs varied modestly in functional avidities for SL9 variants, due to alterations in affinity. This translated to differences in antiviral activities against HIV-1 when functional avidity changes crossed the previously defined threshold required for efficient recognition of HIV-1-infected cells. Higher avidity TCR mutants had generally broader recognition of SL9 variants. Conclusion: These results indicate that rationally targeted increases in functional avidities can be utilized to maximize the antiviral breadth of transgenic TCRs. In contrast to previously reported random mutagenesis to markedly increase functional avidities, tuning through recombining naturally occurring polymorphisms may offer a more physiologic approach that minimizes the risk of deleterious TCR reactivities. © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

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