Volume 8, Issue 4, Pages 530-542 (October 2003) Treatment of rat gliosarcoma brain tumors by HSV-based multigene therapy combined with radiosurgery  Ajay Niranjan, Darren Wolfe, Masakazu Tamura, M Karina Soares, David M Krisky, L Dade Lunsford, Songhui Li, Wendy Fellows-Mayle, Neal A DeLuca, Justus B Cohen, Joseph C Glorioso  Molecular Therapy  Volume 8, Issue 4, Pages 530-542 (October 2003) DOI: 10.1016/S1525-0016(03)00232-6 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Diagrams of recombinant vectors used in this study. Open boxes represent the inverted repeats (TRL/IRL and IRR/TRR) of the HSV genome. Relevant loci are indicated underneath the top diagram and modifications identified in the key at the right. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Southern blot analysis of NUREL-C2. At the top of each part (A–D), a restriction-site map of HSV strain KOS is shown along with the structural organization of the same locus as altered in vector NUREL-C2 and the positions of the respective probes. Functional elements in the NUREL-C2 diagrams are as follows: shaded box (A–C), coding sequence; shaded box (D), ICP4 gene deletion; open arrow, promoter; open hexagon, polyadenylation region; black vertical bar (D), TAATGARAT deletion in the ICP22 and ICP47 promoter regions; open rectangles (D), inverted repeat elements IRL, IRR, and TRR; a′ (D), reiterated a′ element of HSV replication. A–D include a stylized representation of a larger portion of the HSV KOS genome indicating the locus of interest and (Δ) the genome positions replaced by alternate sequences in NUREL-C2; open rectangles represent HSV KOS IR elements. Relevant restriction sites are abbreviated as follows: P, PstI; B, BamHI; R, EcoRI; Bg, BglII; H, HindIII; Sm, SmaI; No, NotI; X, XhoI; S, SalI, Sc, SacI; Bs, BssHI; N, NcoI; En, EcoNI, Hp, HpaI. Underneath, diagnostic Southern blots. Lane numbers are identified above the first blot of each part (C2 for NUREL-C2). Relevant control plasmids included in each blot are described under Materials and Methods. The identities of diagnostic bands are indicated on the right of each blot: W, wild-type HSV (KOS); P, parental vector d106; C2, recombinant vector NUREL-C2. Fragment sizes are indicated on the left of each blot. A (Probes A, B, and C) confirms the structure around the UL23 region of NUREL-C2, which contains a promoter replacement converting UL23 (tk) from an early transcription class to immediate early (IE). Asterisk (*) indicates the endogenous ICP4p loci detected with the ICP4 promoter probe (Probe B). B (probes D, E, F, and G) confirms the insertion of Cx43 driven by the ICP0 promoter and TNFα driven by the HCMV IE promoter into the UL41 locus of NUREL-C2. C (probes H, I, and J) confirms the deletion of the ICP27 locus and the corresponding insertion of the EGFP marker gene driven by the HCMV IE promoter. D (probes K and L) confirms the deletion of ICP4 and the deletion of the TAATGARAT element in the ICP22/47 promoters. Production of viral DNA by high-m.o.i. infection for Southern analysis produces repeated copies of the a′ sequence (a′) responsible for the ladder pattern observed with probe K. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Southern blot analysis of NUREL-C2. At the top of each part (A–D), a restriction-site map of HSV strain KOS is shown along with the structural organization of the same locus as altered in vector NUREL-C2 and the positions of the respective probes. Functional elements in the NUREL-C2 diagrams are as follows: shaded box (A–C), coding sequence; shaded box (D), ICP4 gene deletion; open arrow, promoter; open hexagon, polyadenylation region; black vertical bar (D), TAATGARAT deletion in the ICP22 and ICP47 promoter regions; open rectangles (D), inverted repeat elements IRL, IRR, and TRR; a′ (D), reiterated a′ element of HSV replication. A–D include a stylized representation of a larger portion of the HSV KOS genome indicating the locus of interest and (Δ) the genome positions replaced by alternate sequences in NUREL-C2; open rectangles represent HSV KOS IR elements. Relevant restriction sites are abbreviated as follows: P, PstI; B, BamHI; R, EcoRI; Bg, BglII; H, HindIII; Sm, SmaI; No, NotI; X, XhoI; S, SalI, Sc, SacI; Bs, BssHI; N, NcoI; En, EcoNI, Hp, HpaI. Underneath, diagnostic Southern blots. Lane numbers are identified above the first blot of each part (C2 for NUREL-C2). Relevant control plasmids included in each blot are described under Materials and Methods. The identities of diagnostic bands are indicated on the right of each blot: W, wild-type HSV (KOS); P, parental vector d106; C2, recombinant vector NUREL-C2. Fragment sizes are indicated on the left of each blot. A (Probes A, B, and C) confirms the structure around the UL23 region of NUREL-C2, which contains a promoter replacement converting UL23 (tk) from an early transcription class to immediate early (IE). Asterisk (*) indicates the endogenous ICP4p loci detected with the ICP4 promoter probe (Probe B). B (probes D, E, F, and G) confirms the insertion of Cx43 driven by the ICP0 promoter and TNFα driven by the HCMV IE promoter into the UL41 locus of NUREL-C2. C (probes H, I, and J) confirms the deletion of the ICP27 locus and the corresponding insertion of the EGFP marker gene driven by the HCMV IE promoter. D (probes K and L) confirms the deletion of ICP4 and the deletion of the TAATGARAT element in the ICP22/47 promoters. Production of viral DNA by high-m.o.i. infection for Southern analysis produces repeated copies of the a′ sequence (a′) responsible for the ladder pattern observed with probe K. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Kaplan–Meier survival plot of 9L tumor-bearing rats treated by injection of medium or vector T.1, TH:TNF, or TOCX 3 days post-tumor-cell implantation and daily intraperitoneal GCV administration (days 3–12) with or without GKR performed 5 days post-tumor-cell implantation. SGT, suicide gene therapy mediated by GCV in combination with the HSV-TK enzyme expressed from each vector. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 NUREL-C2 gene expression. Immunoblots demonstrating expression of HSV-TK, connexin43, and ICP0 in NUREL-C2-infected Vero cells. (A) HSV-TK antibody. Vero cells were mock infected (lane 1) or infected with vectors QOZHG (lanes 2 and 3) or NUREL-C2 (lanes 4 and 5) at m.o.i. of 0.3 (lanes 2 and 4) or 3 (lanes 3 and 5). The HSV-tk promoter of QOZHG is inactive in Vero cells due to deletions of the ICP4 and ICP27 genes. (B) Anti-Cx43 antibody. Vero cells were infected with QOZHG (lanes 1–3) or NUREL-C2 (lanes 4–6) at m.o.i. of 0.5 (lanes 1 and 4), 2 (lanes 2 and 5), or 5 (lanes 3 and 6). QOZHG does not carry the Cx43 gene. (C) ICP0 antibody. Vero cells were mock infected (lane 1) or infected with QOZHG (lane 2) or NUREL-C2 (lane 3). ICP0 is the only unaltered IE gene in both vectors. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Kaplan–Meier survival plot of 9L tumor-bearing rats treated by injection of TH:TNF or NUREL-C2 (3 days post-tumor-cell implantation) and daily intraperitoneal administration of GCV (days 3–12) with or without GKR performed 5 days post-tumor-cell implantation. Molecular Therapy 2003 8, 530-542DOI: (10.1016/S1525-0016(03)00232-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions