A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients

Abstract

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral- producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

Autologous, lentivirus‐modified, T‐rapa cell “micropharmacies” for lysosomal storage disorders

“…Fluorometric enzyme assays were modified from previously described methods (Medin et al , 1996a , 1996b ; Okumiya et al , 2006 ; Bedia et al , 2010 ). Substrates used for α‐gal A, GCase, and GAA activities were 5 mM 4‐methylumbelliferyl‐α‐ d ‐galactopyranoside (Sigma‐Aldrich M7633), 15 mM 4‐methylumbelliferyl‐β‐ d ‐glucopyranoside (Sigma‐Aldrich M3633), and 1.5 mM 4‐methylumbelliferyl‐α‐ d ‐glucopyranoside (Sigma‐Aldrich M9766), respectively.…”

Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells

“…Interestingly, a recent report shows 8% of infection of positively selected CD34 + cells, a value identical to the one reported here ( Limon et al , 1997 ). Finally, B cells have been studied so far only using in vitro immortalized EBV‐LCL lines and, in the few reports available, normal HUVEC have shown much lower levels of infection ( Bauer & Miller, 1995; Candotti et al , 1996 ; Hacein‐Bey et al , 1996 ; Medin et al , 1996 ).…”

“…The EGFP gene has already been used in murine bone marrow ( Persons et al , 1997 ) and human cord blood cells ( Verhasselt et al , 1998 ) and shown not to interfere with their in vitro and in vivo differentiating capacity ( Persons et al , 1997 ; Bierhuizen et al , 1997 ; Verhasselt et al , 1998 ). Others have reported alternative selection procedures based on the introduction of membrane proteins ( Bonini et al , 1997 ; Phillips et al , 1996 ; Garcia‐Hernandez et al , 1997 ; Medin et al , 1996 ; Conneally et al , 1996 ; Champseix et al , 1996 ; Su et al , 1997 ). It will be important to test the same vector used here with similar surface markers in order to allow rapid selection of large numbers of infected cells by affinity columns procedures more clinically acceptable than FACS sorting.…”

“…In the few clinical trials with genetically modified cells reported to date, either unselected cells have been injected in patients ( Rosenberg et al , 1990 ; Merrouche et al , 1995 ; Deisseroth et al , 1994 ; Brenner et al , 1993 ; Dunbar et al , 1995 ; Blease et al , 1995 ; Kohn et al , 1995 ; Bordignon et al , 1995 ; Rooney et al , 1995 ; Heslop et al , 1996 ; Onodera et al , 1998 ) or long selection procedures have been applied to the infected cells before administration ( Riddell et al , 1996 ). Ideally one would like to use vectors which confer the infected cells with some marker to allow rapid identification and selection, as reported in a few cases ( Bonini et al , 1997 ; Phillips et al , 1996 ; Garcia‐Hernandez et al , 1997 ; Medin et al , 1996 ; Conneally et al , 1996 ; Champseix et al , 1996 ; Su et al , 1997 ).…”

Protection and selection for gene therapy in the hematopoietic system

“…Membrane antigens have also been investigated as potential in vitro selectable markers. Thus CD24 8, 9, murine heat shock antigen 10, 11 and a truncated version of the nerve growth factor receptor 12 have all been used, along with either FACS or magnetic bead isolation to provide enriched populations of gene‐modified cells. In a similar way, vectors encoding the green fluorescent protein (GFP) or one of its derivatives may also be used.…”