Miskin et al. (RCL assay for EIAV vectors – on line supplementary information) An assessment of vector competition Retroviral vectors can potentially interfere with the replication of another retrovirus, and this has been demonstrated for the murine retroviruses 1. This is usually a result of competition for or interference with cellular receptors. Although it seemed unlikely that VSV-G pseudotyped lentiviral vectors would compete with MLV infection, we wanted to reassure ourselves that this was the case. There is also no evidence that vectors pseudotyped with VSV-G show cross-competition, and it is not anticipated that any other type of envelope could be acquired during human cell production. Therefore, we wanted to test for competition of transduction using very low doses of retroviral or lentiviral vectors with either VSV-G or amphotropic envelopes, in a background of high dose EIAV-VSV-G vector. We reasoned that by measuring transduction using a low dose of vector, any competition observed would predict any potential competition of a similarly coated infectious virus (a hypothetical RCL). To test for vector competition, as a source of background EIAV-VSV-G vector we used a minimal system expressing three genes from the dopamine biosynthetic pathway that we refer to as ProSavin®, and which is expected to enter clinical development for the treatment of Parkinson’s disease 2,3. We then used three vectors that expressed -galactosidase: the first was a minimal EIAV vector pseudotyped with VSV-G (EIAV-LacZ-VSV-G); the second was an MLV vector pseudotyped with VSV-G (MLV-LacZ-VSV-G) and the third was an MLV vector pseudotyped with Amphotropic envelope (MLV-LacZ-Amp). The EIAV-LacZ-VSV-G was used as a model of a hypothetical EIAV-derived RCL with VSV-G. Under RCL assay conditions it would be desirable that the assay could detect a very small dose of an infectious RCL. Therefore we used a minimal dose of the EIAV LacZ-expressing vectors (50 transducing units) that could be reliable counted to model a low dose of RCL in the p1 Miskin et al. (RCL assay for EIAV vectors – on line supplementary information) background of high m.o.i. of EIAV-based test article vector. HEK293 cells were plated in 12-well plates, and the following day were transduced with ProSavin ® at a multiplicity of infection (m.o.i.) of 50 (total of 4.5x10 6 transducing units [TU]); immediately after the addition of ProSavin®, a total of 50 TU of one of the three vectors expressing -galactosidase was added to enable the number of X-gal positive colonies to be counted. The vectors expressing -galactosidase were added within 2 minutes after ProSavin®, and were added sequentially to maximise any potential inhibitory effects of ProSavin® on transduction by the -galactosidase vectors. Replicate transductions were performed without the background ProSavin ® vector for comparison, and all transductions were performed in triplicate. Colonies expressing -galactosidase were counted three days post-transduction following staining with X-gal. As shown in Figure 1, the presence of ProSavin® pseudotyped with VSV-G at an m.o.i. of 50 did not inhibit transduction with either EIAV-LacZ vector or MLV-LacZ vector, irrespective of the envelope glycoprotein used to pseudotype the LacZ vector. There was less than a 2-fold effect between the presence and absence of ProSavin, and statistical analysis using an unpaired 2-tailed T-test indicated that ProSavin did not significantly affect transduction by any one of the three LacZ vectors tested. The reason for the lack of competition is presumably because the VSV-G receptor is not limiting on the target cells. These data indicate that during an RCL assay, infection with a minimal dose of a hypothetical VSV-Gpseudotyped EIAV-based RCL would not be competed-out by the EIAV vector preparation at an m.o.i. of 50, which equates to a 90,000-fold excess. The data also indicate that it is feasible to spike the EIAV vector preparation with MLV without competing with MLV replication, so MLV 4070A would be an appropriate positive control. p2 Miskin et al. (RCL assay for EIAV vectors – on line supplementary information) References 1 Printz M et al. Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays. Gene Ther 1995; 2: 143-150. 2 Azzouz M et al. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease. J Neurosci 2002; 22: 10302-10312. 3 Azzouz M et al. Neuroprotection in a rat Parkinson model by GDNF gene therapy using EIAV vector. Neuroreport 2004; 15: 985-990. Supplementary Figure 1. An assessment of vector competition. A total of 50TU of EIAV or MLV vector expressing -galactosidase (EIAV-LacZ-VSV-G, MLV-LacZVSV-G, or MLV-LacZ-Amp) was added to HEK293 cells either alone, or in the background of EIAV-VSV-G ProSavin® vector at an m.o.i. of 50. The number of X-gal positive colonies that were observed in triplicate wells (±SD) for each condition is shown. The expected number of colonies was approximately 50 (dotted line); the transduction by the marker gene vector varied by less than 2-fold from the expected, whether in the presence or absence of ProSavin®. p3