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Biomedical subjects

A Voytovich

Publications and source records attributed to A Voytovich.

5 recordsLinked to original sources
PubMed1997

RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.

Animals↗
PubMed1996

Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.

Animals↗
PubMed1996

Sustained retroviral gene marking and expression in lymphoid and myeloid cells derived from transduced hematopoietic progenitor cells.

The expression of antiviral genes in human hematopoietic stem or progenitor cells has been proposed as a strategy for gene therapy of AIDS. To be successful, this strategy requires safe and efficient transfer of the therapeutic gene into hematopoietic cells and gene expression has to be maintained in HIV susceptible cells following differentiation. We have used retroviral vectors to transfer the gene for a transdominant inhibitor of HIV replication (RevM10) into CD34+ stem/progenitor cells isolated from human umbilical cord blood (UCB). Following transduction, cells were allowed to differentiate either in vitro in clonogenic assays and long-term stromal cell cultures or in human thymus implanted in immunodeficient scid/scid mice in vivo (SCID-hu). Following differentiation and expansion, multiple lineages of cells were shown to carry the transgene. A higher percentage of gene-marked progenitor cells (10-30% in most cases) were detected in methylcellulose colony assays and in long-term stromal cell cultures (1-5%). In contrast, gene-marked T cells derived from transduced CD34+ cells in a SCID-hu model were detected at an even lower frequency (0.01-1%). RevM10 RNA expression was detected in CD34+ cells immediately after transduction and was maintained after in vitro differentiation of those cells into CD14+ myeloid cells. In T cells, the RevM10-specific RNA was detectable by RT-PCR and also by semiquantitative RNase protection. These findings demonstrate that LTR-driven gene expression is sustained in relevant cells derived from retrovirus-transduced hematopoietic progenitor cells after extensive differentiation in vitro and in vivo and suggest that stringent in vivo, rather than in vitro assays, may be a better preclinical system to improve gene marking and expression in hematopoietic cells.

Acquired Immunodeficiency Syndrome↗