Figure 2
From: AAV2-mediated in vivo immune gene therapy of solid tumours
Validation of immune gene vector construct and transduction efficiency. (a-b) Gene Expression from AAV2-GB. FACS Analysis and ELISA for GM-CSF were used to determine the functionality of AAV2-GB particles in vitro. A 38.2% (+/- 7.4) increase in B7-1 positive cells was observed in AAV2-GB transduced JBS cells. GM-CSF protein was detected in cell culture supernatant in cells transduced with AAV2-GB at a level of 250 pg/ml. (c-e) Transduction of JBS and LLC cells in vitro. The efficiency of AAV2 mediated transduction of the test cell lines JBS and LLC was determined using FACS analysis for cell surface B7-1 expression following AAV2-GB transduction or by luciferase assay following AAV2-Luc transduction. (c) A background level of B7-1 expression of approximately 5% was seen in PBS treated JBS cells while a 13.4% (+/- 0.2) increase in B7-1 positive cells was observed in AAV2-GB transduced JBS cells. (d) A background level of B7-1 expression of 9.4% was observed in PBS treated LLC cells while a 4.25% (+/- 0.15) increase in B7-1 positive cells was observed in AAV2-GB transduced LLC cells. (e) Luminescence was readily detected in both JBS and LLC cells with a significantly higher level evident in JBS cells (p = 0.004). (* Statistical significance (p < 0.05)). (f) Transduction of LLC in vivo with AAV2-GB. A background level of B7-1 expression of approximately 10% was seen in PBS treated LLC cells while a 5.2% (+/- 1.48) increase in B7-1 positive cells was observed in AAV2-GB transduced LLC cells. (g) Transduction of JBS and LLC in vivo with AAV2-Luc. IVIS imaging confirmed AAV transduction of JBS tumours in vivo (9.7 × 10-1 p/sec/cm2/sr/gene copy administered, +/- 0.27) and LLC tumours (4.3 × 10-3 p/sec/cm2/sr/gene copy administered, +/- 0.0009).