Functional and phenotypic characterization of the humanized BLT mouse model.

T cells play a central role in the development of immune responses. Patients lacking T cells because of genetic defects such as DiGeorge or Nezelof syndromes and patients infected with the human immunodeficiency virus are highly susceptible to infections and cancers. The lack of adequate in vivo models of T cell neogenesis have hindered the development and clinical implementation of effective therapeutic modalities aimed at treating these and other clinically important maladies. Transplantation of severe combined immunodeficient (SCID) mice with human hematopoietic stem cells results in long-term engraftment and systemic reconstitution with human progenitor, B, and myeloid cells, but curiously, human T cells are rarely present in any tissue. While the implantation of SCID mice with human fetal thymus and liver (SCID-hu thy/liv mice) allows the development of abundant thymocytes that are localized in the human organoid implant, there is minimal systemic repopulation with human T cells. However, we have recently shown that transplantation of autologous human hematopoietic fetal liver CD34+ cells into the nonobese diabetic (NOD)/SCID mouse background previously implanted with fetal thymic and liver tissues results in long-term, systemic human T cell homeostasis. In addition to human T cells, these mice have systemic repopulation with human B cells, monocytes/macrophages, and dendritic cells (DC). Importantly, in these mice the T cells developed in the human thymic implant are capable of being activated by human antigen-presenting cells and mount potent human MHC-restricted T cell immune responses.

Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.

The exuT gene of Erwinia chrysanthemi EC16: nucleotide sequence, expression, localization, and relevance of the gene product.

Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall. In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration. Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E. chrysanthemi. The GalUA uptake gene (exuT) was cloned and sequenced. Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da. This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile. Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein. This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis. Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA. A targeted mutation in the exuT gene affected the utilization of GalUA as a role carbon source for growth. Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16.