Volume 9, Issue 5, Pages 738-746 (May 2004) Simplified Generation of High-Titer Retrovirus Producer Cells for Clinically Relevant Retroviral Vectors by Reversible Inclusion of a lox-P-Flanked Marker Gene  Rainer Loew, Nathalie Selevsek, Boris Fehse, Dorothee von Laer, Christopher Baum, Axel Fauser, Klaus Kuehlcke  Molecular Therapy  Volume 9, Issue 5, Pages 738-746 (May 2004) DOI: 10.1016/j.ymthe.2004.02.010 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Proviral structure of control and test vectors. (A) The LTRs of control vectors pE178, pE179, and pE188 consist of MPSV-U3 and MMLV-R and -U5 regions followed by Ø, Ø+, and pol/env regions harboring the native MMLV splice donor (SD) and acceptor (SA) sites. The LTRs of control vector pM87o consist of MPSV-U3 and SFFV R- and -U5 regions followed by the MESV Ø region. The eGFP marker gene was introduced with or without lox-P sites into control vectors pE179 and pE178. The transgenes were either truncated CD34 (tCD34, control vector pE188) or C46 (synthetic membrane protein in control vector pM87o). (B) The test vectors were based on either the pE188 or the pM87o backbone and contain the lox-P-flanked marker gene as well as the putative transgene. The resulting test vectors were termed pE189 and pE210. The coding region of the “transgene” is out of frame in both constructs, thus it is not expected to be translated efficiently until removal of the marker gene. (C) The effect of the Cre-recombinase reaction is shown schematically only for pE189. After transduction of the producer cells and selection of the “best” producer cell, the Cre-recombinase expression plasmid (pCMV-Cre) is introduced via transient transfection. Recombinase-mediated excision will then lead to removal of the marker gene together with one of the two lox-P recognition sites from the provirus. The arrows indicate the primer positions for PCR amplification of the proviral region before and after Cre-mediated excision of eGFP. Molecular Therapy 2004 9, 738-746DOI: (10.1016/j.ymthe.2004.02.010) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Purification of PG13 producer pools transduced by control and test vectors and comparison of gene expression levels. (A) Transduced PG13 cell populations were purified either for the marker gene (eGFP) or for the transgene (tCD34, pE188) that was detected via FITC-labeled primary CD34 antibody. To eliminate dead cells from measurements, cells were counterstained with propidium iodide (PI). Gated cells (R1) were used to calculate gene expression level and pool purity. (B) Summarized data of purified pools of all vectors used in the current study. Expression values are given only for green fluorescence. Gene expression of pE188 vector was measured after FITC staining, while all other vectors were measured by eGFP fluorescence. The C46 transgene was stained by PE-labeled secondary antibody for the purification procedure (not shown). Molecular Therapy 2004 9, 738-746DOI: (10.1016/j.ymthe.2004.02.010) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Cre-mediated excision of the marker gene from proviruses of purified PG13-E189 and PG13-E210 producer pools. (A) Result of Cre-recombinase-mediated excision reaction after transient transfection of the purified PG13-E189 producer cells with a Cre expression plasmid. The top shows as a control PG13-E189 cells not transfected with the Cre expression plasmid, while the bottom shows the same cells after transfection. To monitor expression of the transgene (tCD34) the control and transfected cells were incubated with a PE-labeled anti-CD34 primary antibody. The boxed cell fraction was purified as a negative sort (e.g., absence of eGFP fluorescence) and further analyzed. Shown is one representative experiment of three. (B) Effects of eGFP half-life after Cre-mediated excision. Cells were monitored by FACS at days 0, 4, and 7 after transient transfection with the pCMV-Cre plasmid. Cre-mediated excision could be monitored for both producer pools by a long-lasting move of transfected (eGFP-negative) cells toward the PG13 background. Molecular Therapy 2004 9, 738-746DOI: (10.1016/j.ymthe.2004.02.010) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Analysis of PG13-E189 proviral properties. (A) Northern blot analysis of the viral producer cells. The blot was hybridized with a probe directed against the 3′-LTR of the vectors, thus allowing detection of all vector transcripts. The expected sizes of the spliced (*) and unspliced (o) mRNA transcripts are given in the table. (B) PCR analysis of PG13-E189 and PG13-E189.fl proviruses. Genomic DNA was prepared from purified cell populations. In test reactions (T), the expected fragments before (left panel) and after (right panel) eGFP excision were amplified, while all controls containing either only the sense or only the antisense primer with template (K1, K2) or both primers but no template (K3) gave no signal. (C) Sequence analysis of cloned PG13-E189.fl PCR fragment. The sequence shown was identical in five of five clones analyzed. The lox-P site is underlined and only the fusion point adjacent sequence stretches are given. Molecular Therapy 2004 9, 738-746DOI: (10.1016/j.ymthe.2004.02.010) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 General outline of viral producer clone selection. The viral genome is introduced into a producer cell by infection, at low m.o.i. (<0.5), ensuring single-copy integration of the vector to be produced. (A) The simplest selection process for a viral producer clone is described. The transgene allows direct or antibody-based selection of transduced cells and 50–100 of those are selected and tested for titer production. From the best producer clone a primary seed bank is established. (B) The steps necessary for establishing a producer clone for a therapeutic vector carrying a transgene that cannot be readily detected are described. Therefore the initial cloning step is not directed but “blind” and requires cultivation of 1000–10,000 individual clones to obtain the necessary amount of 50–100 positively transduced producer cells. Because detection of positive clones is possible only by intracellular methods, the cells need to be fixed or prepared for the respective testing, but cannot be used for further cultivation, which makes it necessary to split each individual clone before the detection procedure can be started. Titration of the viral supernatants produced by the selected positive cells requires the same treatment. (C) Simplified establishment of viral producer clones for vectors described in (B). The marker gene flanked by recombinase recognition sites has to be introduced into the viral vector prior to transduction of packaging cells. The marker gene allows direct detection of producer cells and only 50–100 clones have to be tested to isolate the “best.” Into the final clone the recombinase is transiently introduced (either as protein or encoded on an expression plasmid). After the recombinase reaction cells are monitored for the absence of the marker gene. At this point it has to be decided whether an additional subcloning step should be done or if the resulting cells are expanded directly for the primary seed bank. The additional steps necessary are underlined. Molecular Therapy 2004 9, 738-746DOI: (10.1016/j.ymthe.2004.02.010) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions