1. Volume 14, Issue 5, Pages 735-744 (November 2006)
Targeted retroviral vectors displaying a cleavage site-engineered hemagglutinin (HA) through HA–protease interactions 
Judit Szécsi, Rosybel Drury, Véronique Josserand, Marie-Pierre Grange, Bertrand Boson, Irene Hartl, Richard Schneider, Christian J. Buchholz, Jean-Luc Coll, Stephen J. Russell, François-Loïc Cosset, Els Verhoeyen 
Molecular Therapy 
Volume 14, Issue 5, Pages (November 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Characterization of vector particles pseudotyped with FXa substrate-displaying glycoproteins. MLV vector particles carrying a GFP marker gene and harboring the wild-type gps derived from MLV, Ebola virus, and influenza virus (wt) or carrying the corresponding gps modified by insertion of a Factor Xa (Xa) substrate (IEGR sequence, in one-letter amino acid code) inserted between their surface and transmembrane subunits, were produced by transient transfection of 293T cells. For Factor Xa treatment of vector particles, viral supernatants were incubated with purified FXa protease (Promega) at 4 mg/ml and CaCl2 (2.5 mM) for 60 min at 37°C. (A) Viral supernatants treated (+), or not treated (−), with purified recombinant FXa were used to transduce TE671 (for vectors pseudotyped with MLV gp and HA) or Vero (for vectors pseudotyped with Ebola virus gp) target cells. The transducing titers, expressed as infectious units, are the means ± standard deviation of five independent experiments. The arrows indicate the titers that were below the background levels of detection. Due to the use of a FACS method to monitor transduced cells, the determination of GFP-positive cell below 0.1%, i.e., corresponding to infectious titers below 103 iu/ml under our experimental conditions, could not be accurately determined. The background level of transduction was therefore fixed at 103 iu/ml, by using noninfectious vector particles devoid of gp. (B) The purified vector particles were analyzed by Western blot before and after treatment with FXa using antibodies against the different gps. SU, surface subunit of MLV gp; GP0, precursor of Ebola virus GP; GP2, transmembrane subunit of Ebola virus GP; HA0, HA precursor; HA1, HA surface subunit; HA2, HA transmembrane subunit. The positions of the molecular weight markers are shown (kDa).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Characterization of recombinant hemagglutinins harboring MMP-modified cleavage sites. (A) The sequence upstream to the multibasic KKRKKR cleavage site of the wild-type HA (HA wt) was modified to create a BstBI restriction site, allowing replacement of the HA cleavage site with alternative protease substrates. The following substrate sequences were introduced (underlined): the Factor Xa cleavage site (HA–Xa), an MMP-2 substrate (HA–MMP2), and three newly identified MMP substrates (HA–AK, HA–PQ, and HA–RS). The arrows indicate the potential position of cleavage for each substrate sequence. (B) Western blot analysis of purified vector particles incorporating the wt HA or the modified HA chimeras using antibodies against HA or MLV capsid protein. HA0, HA precursor; HA1, HA surface subunit; HA2, HA transmembrane subunit. Control vector particles were generated without gp (no gp) or with HA-Ct, a recombinant HA containing a noncleavable linker. The positions of the molecular weight markers are shown (kDa). (C) Transducing titers of vector particles carrying wt HA, control HA, or MMP-modified HA chimeras determined on MMP-poor TE671 target cells. The transducing titers, expressed as infectious units/ml, are the means ± standard deviation of five independent experiments. The arrows indicate the titers that were below the background levels of detection.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Expression of membrane-type MMPs in cells producing HA-modified vector particles. Vectors displaying wt HA (HA wt), control HA (HA–Ct), or HA chimeras were produced in the absence (−) or in the presence of two membrane-type MMPs, MT1-MMP (MT1) or MT2-MMP (MT2), upon expression in producer cells. (A) Western blot analysis of purified vector particles incorporating the wt or chimeric HA using antibodies against HA or the MLV capsid protein. HA0, HA precursor; HA1, HA surface subunit; HA2, HA transmembrane subunit. The positions of the molecular weight markers are shown (kDa). (B) Transducing titers of vector particles produced in the absence (no MT-MMP) or in the presence of MT1-MMP (+ MT1-MMP) or MT2- MMP (+ MT2-MMP), as determined on MMP-poor TE671 target cells. The transducing titers, expressed as infectious units, are the means ± standard deviation of five independent experiments. The arrows indicate the titers that were below the background levels of detection.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Vector particles displaying MMP-modified HA chimeras efficiently transduce MMP-rich target cells. (A) Transducing titers of vector particles carrying the wt HA, control HA (HA–Ct), or HA chimeras on MMP-poor TE671 cells and MMP-rich HT1080, U118, U87, and U251 cells. The transducing titers, expressed as infectious units, are the means ± standard deviation of five independent experiments. The arrows indicate the titers that were below the background levels of detection. (B) Average fold increase in transducing titers on MMP-rich cells relative to titers on MMP-poor TE671 cells. The differences in intrinsic permissivity to transduction of the different target cells were normalized using the titers obtained with vector particles carrying the wt HA. (C) U251 target cells were transduced in the presence of different concentrations of 1,10-phenanthroline (Aldrich), an MMP inhibitor (12.5 or 50 μM). The results show the percentage inhibition of transduction levels obtained with 1,10-phenanthroline relative to those of the same vector particles in the absence of inhibitor.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Vector particles harboring MMP-modified HA chimeras allow selective gene transfer into MMP-expressing xenografts in nude mice. MMP-poor TE671 and MMP-rich U251 cells were injected sc into nude mice that developed solid tumors ca. 14 days later. Vector particles pseudotyped with wt HA or HA–AK chimeras were injected into the xenografts. (A) In vivo bioluminescence imaging of xenografts after injection of luciferase substrate are shown for each mouse. A bioluminescence scale ranging from 2000 to 5050 relative light units (RLU) was applied. (B) Determination of luciferase activity, expressed as RLU/mg protein, in cells extracted from TE671 or U251 xenografts transduced with vector particles harboring wt HA or HA–AK chimera or with control vector particles. The values are the means ± standard deviation of four independent xenografts for each condition.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Selective gene delivery to MMP-rich cells in mixed cell populations. MMP-poor TE671 cells were labeled by the red fluorescent protein (RFP) marker gene. RFP-labeled TE671 cells were mixed with unlabeled MMP-rich U87 or U251 target cells. The mixed cell populations were transduced with vector particles carrying wt HA or MMP-modified HA chimeras and a GFP marker gene. (A) Two-color FACS analysis of the heterogeneous population containing MMP-poor RFP(+) and MMP-rich RFP(−) cells after transduction with vectors carrying the indicated gp. The percentage of transduced cells in each quadrant is indicated. (B) Index of selectivity determined as the ratio of transduced GFP(+) MMP-rich U87 or U251 cells to MMP-poor TE671 cells, following transduction in mixed cell populations. The results of three independent experiments are shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions