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
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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.
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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®.
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