Fig. 5.
CD22-RTM abrogates the ability of leukemia-initiating ALL xenograft cells derived from B-lineage ALL patients to cause leukemia in NS mice.
B-lineage ALL xenograft cells (2 × 106 cells/sample) were transfected with 5 μg CD22-RTM plasmid DNA using the Amaxa Nucleofector Kit and then inoculated into NS mice (N = 17) intravenously via tail vein injections in a 200 μL total volume. Controls included NS mice inoculated with untransfected xenograft cells (N = 5), xenograft cells transfected with 5 μg empty plasmid (EPL) (N = 17) or xenograft cells transfected with 5 μg Dystrophin-RTM (N = 5) prior to inoculation. Mice were monitored for signs of leukemia and all mice in a given experiment were sacrificed when any mouse developed morbidity. (A–C) RT-PCR analyses of xenograft clone #1 transfected with CD22-RTM vs. control plasmids confirming transfection and successful completion of trans-splicing. Total RNA for the PCR was extracted 2 days after transfection. CON: untransfected cells. (A) An agarose gel is depicted showing the RT-PCR amplification of a 415-bp RTM-specific RNA-segment amplified by RT-PCR using a CD22 Exon 11 primer as forward primer (EX11-F) and the RTM IRES primer RTM-4494-5263 as a reverse primer (IRES-R). The detection of the amplicon provides evidence for the successful transfection of ALL xenograft cells with the CD22-RTM. (B) Successful completion of selective CD22 RTM-induced trans-splicing in B-lineage ALL xenograft cells was evidenced by specifically amplifying a 763-bp trans-spliced mRNA segment using an Exon 9 forward primer (EX9-F) and a reverse primer in the IRES sequence of the RTM (IRES-R), as previously reported (Uckun et al., 2015a). (C) An agarose gel of the RT-PCR products obtained with the EX4-F/EX4-R primer set that was used as a positive control of RNA integrity is shown. (D and E) The cumulative results for the average spleen size and nucleated spleen cell count are shown for each treatment group, respectively. (F) Spleen images from representative mice are shown. The spleen images were obtained using an iPhone 4S equipped with an 8-megapixel iSight camera.