Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential.

To examine the role of the Notch ligand Delta-4 on hematopoietic stem cells, human CD34+CD38low cord blood cells were cocultured on S17 cells transduced with transmembrane Delta-4 (mbD4/S17) or an empty vector (C/S17). By the end of a 3-week culture, mbD4/S17 induced a 25-fold reduction in nucleated cell production, as compared to C/S17, by maintaining a higher proportion of cells in G0/G1 phase. A specific retention of a high proportion of CD34+ cells throughout the culture was observed with mbD4/S17, contrary to C/S17. Although mbD4/S17 promoted expansion of cells with the phenotype of committed lymphoid precursors (CD34+CD7+CD45RA+), these cells still retained their myeloid differentiation potential. mbD4/S17 maintained a higher LTC-IC frequency in output CD34+ cells, compared to C/S17, as in the subsets of cells having completed the same number of divisions on mbD4/S17. A Delta4-Fc protein (extracellular part of human Delta4 fused to Fc human IgG1 portion), immobilized on plastic, also reduced cell production and retained the LTC-IC potential. Transplantation of cells grown on mbD4/S17 into NOD/SCID mice showed no significant enhancement of the long-term repopulating ability. Thus, Delta4 appears to inhibit hematopoietic stem cell proliferation, in association with the maintenance of short-term lymphoid and myeloid repopulation capacity.

A reassessment of the MAdCAM-1 structure and its role in integrin recognition.

Mucosal addressin cell-adhesion molecule (MAdCAM-1) is a membrane-bound leukocyte receptor regulating both the passage and retention of leukocytes in mucosal tissues. A crystal structure for the two extracellular amino-terminal domains of human MAdCAM-1 has previously been reported, confirming their expected immunoglobulin superfamily topology. In this study, a second crystal structure of this fragment is described. Although the overall structure is similar to that previously reported, one edge strand in the amino-terminal domain is instead located on the opposite sheet. This alters the arrangement and conformation of amino acids in this region that have previously been shown to be crucial for ligand binding. MAdCAM-1 is also seen to form dimers within the crystal lattice, raising the possibility that oligomerization may influence the biological role of this adhesion molecule.

A novel human packaging cell line with hematopoietic supportive capacity increases gene transfer into early hematopoietic progenitors.

The hematopoietic stem/progenitor cell (HSPC) represents the ideal target for gene therapy of disorders of the hematopoietic system, but still faces problems related to ex vivo manipulation and gene transfer efficiency. We demonstrate that soluble factors from the human endothelial-like cell line ECV 304/T24 support the growth of human CD34(+) progenitor cells as primary human bone marrow stroma and increase the rate of gene transfer into progenitor cells up to 5-fold. ECV 304/T24 was used to generate split-function amphotropic packaging cell lines (named APEX) with the purpose of combining, in the same cells, hematopoietic support and gene transfer vehicle functions. The APEX cell lines were negative for the presence of replication-competent retroviruses and produced complement-resistant vector particles. When mobilized peripheral blood or umbilical cord blood CD34(+) cells were exposed once to APEX supernatants, the level of gene transfer was equivalent to that observed with GP + Am12, in spite of the lower titer of the APEX producers. More importantly, APEX supernatants gave rise reproducibly to a 2-fold increase in transduction of early progenitors (long-term culture-initiating cells), reaching on average 50% gene transfer. This novel packaging cell represents a significant advance in HSPC genetic modification technology, combining both a beneficial hematopoietic supportive effect and the gene transfer vector function in a human-based system.

Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells.

BACKGROUND: Clinically applicable protocols for ex vivo modification of human CD34+ hematopoietic stem/progenitor cells rely on incubation of the target cell with supernatant containing recombinant retroviral particles. Although components of the supernatant may have a profound impact on both preclinical and clinical outcome, to date supernatant production has not been properly addressed with regard to CD34+ cells. We wanted to investigate and optimise production conditions for this target using simple, reproducible and clinically applicable procedures and reagents. METHODS: Retroviral supernatant was obtained from producer cell GP+Am12 under various production conditions and tested for bulk transduction efficiency and endpoint titre on murine and human cell lines. Gene transfer efficiency into CD34+ cells from mobilised peripheral blood, after a single exposure to retroviral supernatant, was measured by transgene expression, colony forming assay and long-term culture colony forming assay. RESULTS: Bulk gene transfer or endpoint titre values obtained on cell lines for the different production conditions were not predictive of gene transfer efficiency into hematopoietic progenitors. Time of virus production appeared to have the greatest impact on gene transfer, peaking at 6 h and decreasing 2-3-fold at longer time points. Neither the culture vessel used nor the temperature for virus production had any significant effect on gene transfer into CD34+ cells. Supernatant could be produced under defined serum-free conditions as efficiently as serum containing conditions for CD34+ cell gene transfer. CONCLUSIONS: The present data provide important implications for the establishment of quality controls for small- and large-scale clinical grade supernatant production for gene transfer into human hematopoietic stem/progenitor cells.

Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer.

We report increased transduction of human hematopoietic progenitor cells through a combination of novel retroviral vector packaging cell lines, and improved vector supernatant production. The new ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic particles (ProPak-A cells), and ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing. Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells. All vector types transduced primary human CD34-positive or CD4-positive cells, regardless of tropism. However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector. The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines. This ping-pong amplification yielded supernatant containing vector targeted to two distinct receptors present on human cells, and did not result in detectable RCR formation. In addition, we describe conditions for improved vector supernatant production in a packed-bed bioreactor.

Improved detection of replication-competent retrovirus.

Recombinant retroviral vectors are the predominant delivery system in human gene therapy protocols. Since contaminating replication-competent retrovirus (RCR) can arise during the production of retroviral vector supernatants, sensitive assays for the screening of supernatants are necessary. In this study, we present a marker rescue assay based upon a Mus dunni cell line stably transduced with a lacZ gene. We show that detection of RCR in vector supernatants by the M. dunni lacZ marker rescue assay or PG-4 S+ L- focus-forming assay is equally sensitive. By inoculating test supernatants under centrifugation (which we term spinoculation), we increased the sensitivity of detection of RCR 10 to 100-fold with the PG-4 S+ L- and lacZ marker rescue assays. While the spinoculation protocol had no adverse effects on cells, spinoculation of high titer vector supernatants onto PG-4 cells resulted in some cytotoxicity, making identification of RCR positive cultures difficult. However, spinoculation of vector supernatants onto M. dunni lacZ cells resulted in no cytotoxicity, and also partially overcame inhibition of detection of low levels of RCR due to the presence of high titer replication-incompetent vector.

A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety.

Successful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in vivo gene delivery.

Detection of intracellular HIV-1 Rev protein by flow cytometry.

The Rev trans-activator protein plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) replication by allowing expression of the viral structural proteins. We have developed a protocol to quantitatively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabilization techniques were compared. These protocols varied in the magnitude of the signal which could be detected, and in the ability to distinguish between Rev Ag positive and negative populations. This technology is applicable to a variety transduced or transfected cell types (species, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize delivery of the dominant-negative RevM10 gene for clinical gene therapy applications. In addition, a second, independent intracellular protein (HIV-Tat) has been detected using the same approach.