Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cells

Abstract

Background Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed. Methods In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition. Results Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. Conclusions Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification.

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice

“…However, it is difficult to predict how these LVs will behave in vivo. 32 One study performed intrafemoral injection of murine retroviral vectors into Jak-3 knockout mice. 33 That study, however, used 5-fluorouracil preconditioning, which induces cycling and possibly differentiation of the very primitive early progenitors.…”

Retargeting Vesicular Stomatitis Virus Glycoprotein Pseudotyped Lentiviral Vectors with Enhanced Stability byIn SituSynthesized Polymer Shell

“…These approaches have not been generally applicable because modifications in native envelope lead to large reductions in viral titer, and pseudotypes with other viral envelopes are not generally applicable to targeting many different types of cells and tissues (Kasahara et al, 1994;Valsesia-Wittmann et al, 1994;Han et al, 1995;Somia et al, 1995;Marin et al, 1996;Nilson et al, 1996;Yu and Schaffer, 2005). We previously developed and described a targeting lentiviral vector pseudotyped with a modified version of the Sindbis virus envelope proteins that can target human leukocyte antigen (HLA) class I, CD4, CD19, CD20, CD45, CD146, CD34, P-glycoprotein of melanoma cells, and prostate stem cell antigen either in vitro or in vivo (Morizono et al, 2001(Morizono et al, , 2006Morizono and Chen, 2005;Pariente et al, 2007Pariente et al, , 2008Liang et al, 2009aLiang et al, , 2009b. The distinguishing properties of this vector relative to past retroviral targeting vectors were that it could be produced in high titers and home to specific cells and tissues after systemic administration via the bloodstream.…”

“…First, modifications to vectors must be made so that they can utilize unique cell surface molecules as new receptors to redirect vector binding to the desired target cells. We have successfully accomplished targeted transduction in vitro and in vivo using a modified Sindbis virus envelope pseudotype (Morizono et al, 2001(Morizono et al, , 2006(Morizono et al, , 2009(Morizono et al, , 2010Pariente et al, 2007Pariente et al, , 2008Liang et al, 2009b). Our initial construct consisted of a Sindbis virus envelope pseudotype modified by conjugation with affinity reagents such as antibodies directed to cell surface molecules or genetically engineered for covalent incorporation of ligands that bind specific cell surface molecules.…”

Specific Transduction of HIV-Susceptible Cells for CCR5 Knockdown and Resistance to HIV Infection: A Novel Method for Targeted Gene Therapy and Intracellular Immunization

“…These engineered vectors have been shown to transduce specific cell types such as CD34 + cord blood stem cells, metastatic melanoma cells, tumor cell lines, and cells expressing CD4 or the human leukocyte antigen. 42-47 …”