Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is protective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the CNS when delivered peripherally is limited due to the blood-brain barrier. In the present study, bone marrow cells were used as vehicles to deliver the BDNF gene into the CNS.

Protection of nigral neurons by GDNF-engineered marrow cell transplantation.

Marrow stromal cells, which have many characteristics of stem cells, populate various non-hematopoietic tissues including the brain. In the present study, the cDNA for the dopaminergic neurotrophic factor Glial Cell Line-Derived Neurotrophic Factor (GDNF) was delivered using marrow cells in the mouse 1-Methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) model of Parkinson's disease. Following cross-sex intravenous bone marrow transplantation with male donor cells that had been transduced with GDNF (GDNF-BMT) or with non-manipulated marrow (Control-BMT), female recipient mice were subjected to systemic MPTP injections.

Enhanced vulnerability to oxidative stress by alpha-synuclein mutations and C-terminal truncation.

alpha-Synuclein is a key component of Lewy bodies found in the brains of patients with Parkinson's disease and two point mutations in this protein, Ala53Thr and Ala30Pro, are associated with rare familial forms of the disease. Several lines of evidence suggest the involvement of oxidative stress in the pathogenesis of nigral neuronal death in Parkinson's disease. In the present work we studied the effects of changes in the alpha-synuclein sequence on the susceptibility of cells to reactive oxygen species.

Targeting of marrow-derived astrocytes to the ischemic brain.

Bone marrow progenitor cells have been shown to contribute to a small proportion of cells in nonhematopoietic tissues including the brain. In the acute unilateral middle cerebral artery occlusion model in spontaneously hypertensive rats following male-to-female bone marrow transplantation, we present data suggesting that 55% more marrow-derived cells, in general, and 161% more GFAP-positive astrocytes, in particular, migrate preferentially to the ischemic cortex than to the contralateral non-ischemic hemisphere. In addition to their biological significance, our findings could have therapeutic implications.

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses.

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth.

Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice.

Glial cells are thought to derive embryologically from either myeloid cells of the hematopoietic system (microglia) or neuroepithelial progenitor cells (astroglia and oligodendrocytes). However, it is unclear whether the glia in adult brains free of disease or injury originate solely from cells present in the brain since the fetal stage of development, or if there is further input into such adult brains from cells originating outside the central nervous system. To test the ability of hematopoietic cells to contribute to the central nervous system, we have transplanted adult female mice with donor bone marrow cells genetically marked either with a retroviral tag or by using male donor cells.

Transduction of human hematopoietic progenitor cells with retroviral vectors based on the gibbon ape leukemia virus.

Gene transfer into human hematopoietic stem cells continues to be complicated by issues of transfer efficiency. We have examined the capacity of newly described retroviral vectors based on the gibbon ape leukemia virus (GaLV) to introduce genes into human hematopoietic progenitor cells. Total nucleated human bone marrow cells were transduced using GaLV vectors packaged with either amphotropic or GaLV envelopes.

Evaluation of retroviral vectors based on the gibbon ape leukemia virus.

The gibbon ape leukemia viruses (GaLVs) are primate-derived C-type retroviruses with a broad host range. Using an infectious, full-length clone of the GaLV SEATO strain, we have determined that this virus replicates efficiently in 13 of 17 human cell lines tested. In fact, the SB lymphoblast cell line, while resistant to infection by wild-type amphotropic mouse leukemia virus (A-MLV), was infected by GaLV-SEATO.

Gibbon ape leukemia virus and the amphotropic murine leukemia virus 4070A exhibit an unusual interference pattern on E36 Chinese hamster cells.

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV.

Factors affecting retroviral vector function and structural integrity.

Recombinant retroviruses are widely used for gene transfer into eukaryotic cells and exhibit significant potential for human gene therapy. Despite the utility of retroviral vectors, their design is still essentially empirical. We have constructed a series of reciprocal, double-gene vectors to compare the dual expression of beta-galactosidase (beta-gal) and neomycin phosphotransferase (neor) in a retroviral delivery system.

Introduction of human genomic sequences renders CHO-K1 cells susceptible to infection by amphotropic retroviruses.

To learn more about the nature of the block to infection by amphotropic retroviruses exhibited by Chinese hamster cells (CHO-K1), CHO-K1 cells were made susceptible to amphotropic retrovirus infection by introducing genomic DNA from infectable human cells. A clone, designated CHO18, was obtained and shown to be infected as efficiently as NIH 3T3 fibroblasts. Susceptibility of CHO18 cells to infection was specific to retroviruses and vectors bearing an amphotropic envelope.

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L.

Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow.

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony-forming unit granulocyte-macrophage after more than 200 days.

Positive selectable markers for use with mammalian cells in culture.

As more complicated gene expression studies are necessary, the need for multiple positive selection schemes becomes critical. Numerous selectable markers have been described over the last 25 years. A hallmark of the most generally useful markers is easy selection in a wide number of cell types.

Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer.

In multidrug resistance, cells become simultaneously resistant to anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain other natural product cytotoxic drugs. Resistance results from synthesis of a multidrug transporter (P-glycoprotein) encoded by the MDR1 gene (also known as the PGY1 gene). In the present study, a retrovirus vector containing a complementary DNA for the human multidrug resistance gene HaMDR1/A was used to transfer the multidrug resistance phenotype to bone marrow cells of the DBA/2J mouse.

Increased efficiency of gene transfer with retroviral vectors in neonatal hematopoietic progenitor cells.

The retroviral vector N2, which is derived from the Moloney murine leukemia retrovirus, was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells from fetal, neonatal, and adult dogs and cats. Infection of canine and feline bone marrow cells with the N2 vector resulted in resistance of granulocyte-macrophage colony-forming units (CFU-GM) to G418. Approximately 2%-4% of fetal liver, fetal bone marrow, and adult bone marrow day-7 CFU-GM were resistant to 1.

In utero gene transfer and expression: a sheep transplantation model.

Retroviral-mediated gene transfer was used to insert a Neo R gene into fetal sheep hematopoietic cells obtained by exchange transfusion from lambs in utero. After gene transfer the cells were returned to the donor fetus. The lambs were examined after birth for the presence of a functioning Neo R gene.

Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using retroviral vectors.

The Moloney murine leukemia retrovirus-derived vector N2 was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells. Approximately 5% of day seven CFU-GM were resistant to 2,000 micrograms/ml G418, using a supernatant infection protocol in the absence of vector-producing cells. A greater proportion of CFU-GM colonies were recovered relative to uninfected controls as the stringency of selection was diminished.

Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype.

In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells.

Retroviral-mediated gene transfer into hemopoietic cells.

Retroviral vectors have provided a means for the introduction of functioning exogenous genes into the hematopoietic system of whole animals. Although these vectors are quite efficient in the mouse model, when applied to non-murine in vivo systems, the efficiency of gene transfer has diminished to impractical levels. Since in vivo analyses are expensive and time consuming, in vitro models have been developed to speed the evaluation of alternative protocols.

Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

Gene therapy: efforts at developing large animal models for autologous bone marrow transplant and gene transfer with retroviral vectors.

Two new large animal models, non-human primates and fetal sheep, have been developed in an effort to determine the feasibility of using retroviruses for gene therapy. The retroviral vectors N2 and SAX have been used to introduce the genes for neomycin phosphotransferase (neoR, conferring resistance to the antibiotic G418) and human adenosine deaminase (ADA; EC 3.5.

Retroviral-mediated gene transfer into mammalian cells.

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells.