Biomedical subjects

Karen Pollok

Publications and source records attributed to Karen Pollok.

5 recordsLinked to original sources

PubMed2006

G2 cell cycle arrest and cyclophilin A in lentiviral gene transfer.

Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34(+) peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, rather than PTK inhibition, appears to be the major factor responsible for increased gene transfer. Genistein also increases cyclophilin A (CypA) protein, a cellular protein important for efficient HIV-1 infection. While we show that CypA(-/-) Jurkat cells transduce poorly with lentiviral vectors, genistein does increase gene transfer in CypA-deficient cells. CypA and G2 cell cycle arrest appear to be two independent factors important for efficient lentiviral gene transfer. The role of genistein and other G2-arresting agents may be useful for improving the efficiency of lentiviral gene therapy.

Animals↗

PubMed2005

Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions.

HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies.

Antigens, CD34↗

PubMed2004

Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.

Emerging evidence to support the use of endothelial progenitor cells (EPCs) for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression is compelling. However, there is no uniform definition of an EPC, which makes interpretation of these studies difficult. Although hallmarks of stem and progenitor cells are their ability to proliferate and to give rise to functional progeny, EPCs are primarily defined by the expression of cell-surface antigens. Here, using adult peripheral and umbilical cord blood, we describe an approach that identifies a novel hierarchy of EPCs based on their clonogenic and proliferative potential, analogous to the hematopoietic cell system. In fact, some EPCs form replatable colonies when deposited at the single-cell level. Using this approach, we also identify a previously unrecognized population of EPCs in cord blood that can achieve at least 100 population doublings, replate into at least secondary and tertiary colonies, and retain high levels of telomerase activity. Thus, these studies describe a clonogenic method to define a hierarchy of EPCs based on their proliferative potential, and they identify a unique population of high proliferative potential-endothelial colony-forming cells (HPP-ECFCs) in human umbilical cord blood.

Blood Cells↗

PubMed2004

Evaluation of plasmid DNA removal from lentiviral vectors by benzonase treatment.

To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37 degrees C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 +/- 0.3 x 10(6) transduction units per milliliter (TU/ml) and 2.7 +/- 0.3 x 10(6) TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

Adult↗

PubMed2003

A theoretically optimized method for cord blood stem cell cryopreservation.

The objective of this study was to develop an optimal cryopreservation method for human umbilical cord blood hematopoietic progenitor cells as evidenced by improved retention of in vivo engraftment ability and multilineage differentiation. An extended understanding of the osmometric/permeability characteristics of cord blood stem cells was accomplished by measuring permeability of the cryoprotectant dimethyl sulfoxide (DMSO) at below-ambient temperatures (10 degrees and 3 degrees C). These data were combined with previously published osmotic and permeability data and the water-NaCl-DMSO phase diagram in conjunction with a mathematical model to determine an optimal initial DMSO concentration, cooling rate, and liquid nitrogen plunging temperature. Cells cryopreserved with the theoretically optimized procedure were then compared with cells frozen using standard methods for the ability to engraft in irradiated NOD/SCID mice. The optimal procedure was determined to include a 0.7 molal (approximately 5%) DMSO concentration at a cooling rate of 4 degrees C/min, and a plunging temperature of -44 degrees C. The optimized protocol resulted in significantly higher engraftment of human CD45(+) cells (17.2 +/- 1.6% vs. 8.4 +/- 1.6%), CD19(+) B lymphocytes (11.3 +/- 1.2% vs. 5.8 +/- 1.2%), and CD34(+) cells (1.9 +/- 0.09% vs. 0.6 +/- 0.09%) compared to cells frozen using a standard method. Engraftment of CD33(+) cells was not significantly different (4.0 +/- 0.3 vs. 3.2 +/- 0.6, respectively). This study demonstrated that the use of a theoretically determined optimal cryopreservation method is superior to standard methods for maintaining UCB PCBs with multilineage repopulation potential in NOD/SCID mice.

Antigens, CD↗