Volume 15, Issue 11, Pages 1973-1981 (November 2007) A Novel Dual-targeted Lentiviral Vector Leads to Specific Transduction of Prostate Cancer Bone Metastases In Vivo After Systemic Administration  Nonia Pariente, Kouki Morizono, Mandeep S Virk, Frank A Petrigliano, Robert E Reiter, Jay R Lieberman, Irvin SY Chen  Molecular Therapy  Volume 15, Issue 11, Pages 1973-1981 (November 2007) DOI: 10.1038/sj.mt.6300271 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Lentiviral vectors carrying the PSE-BC promoter drive tissue-specific transcription in vitro. For all cell types, 1 × 105 cells were infected with FUGW or FPGW pseuotyped with vesicular stomatitis virus glycoprotein. FUGW carries the ubiquitin-C (Ubi-C) promoter driving the expression of enhanced green fluorescent protein (EGFP), FPGW is the same vector but with the PSE-BC promoter. EGFP expression was analyzed 3 days after transduction. The percentage of infectivity and mean fluorescence intensity of the EGFP-positive population are indicated in each plot. The gates were set using parallel mock infections. LPSCA4 is a cell line obtained after stably expressing the prostate stem cell antigen (PSCA) in LNCaP cells. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Lentiviral vectors carrying the PSE-BC promoter drive tissue-specific expression in vivo. Four male severe combined immunodeficiency (SCID) mice were injected with 1 × 106 (a) 293T or (b) LAPC-9 cells in the left flank. When the tumors reached 0.5 cm in diameter, vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors carrying the ubiquitin-C (Ubi-C) or the PSE-BC promoter driving firefly luciferase (FUhLucW or FPhLucW) were injected intratumorally. P/s/cm2/sr is photons/second/cm2/steridian. (a) 293T tumors. Two representative animals from each group are shown. Luciferase expression was analyzed 14 days after virus injection. Maximum photon emission values for the FUhLucW group were 3.9 × 108 and 2.6 × 108 p/s/cm2/sr, and for the FPhLucW group 1.4 × 105 and 2.5 × 105. (b) LAPC-9 tumors. Luciferase expression values 7, 14, and 25 days after vector injection are shown for one representative animal from each group. Maximum photon output for the animal injected with FUhLucW was 5.3 × 107, 5.5 × 107 and 5.3 × 107 p/s/cm2/sr on days 7, 14, and 25, respectively, and for the animal injected with FPhLucW, 6.9 × 106, 1.6 × 107, and 5.9 × 107. (c) Naïve SCID mice were injected through the tail vein with FUhLucW or FPhLucW (VSV-G) mixed with FUIntronRW (VSV-G) a vector expressing renilla luciferase under the control of the Ubi-C promoter. Two representative animals from each group are shown. The top panels present firefly luciferase expression measured 16 days after vector injection (the maximum photon output for the animals injected with FUhLucW was 2.5 × 106 and 1.3 × 106 p/s/cm2/sr while for the FPhLucW group it was 9.3 × 103 and 1.6 × 104). The lower panels show renilla luciferase detection in the same animals 15 days after vector injection. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 1G8 antibody can mediate specific targeting to prostate stem cell antigen (PSCA)-expressing cells. 1 × 105 LNCaP or LPSCA4 cells were infected with 2.2 pseudotypes of FPGW in the absence of antibody or with of 1G8 or αHLA antibody (indicated on the left of the panels). Human leukocyte antigen (HLA) is expressed in both cell types, whereas PSCA is expressed only in LPSCA4 cells. Enhanced green fluorescent protein (EGFP) expression was determined 3 days after transduction. The percentage of EGFP positive cells and mean fluorescence intensity is indicated in each plot. The gate was set using a parallel mock infection. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Double targeting of lentiviral vectors synergistically enhances specificity in vitro. (a) 1 × 105 LPSCA4 or HepG2 cells were infected with FPGW (2.2), FUGW (2.2) or FPGW (vesicular stomatitis virus glycoprotein (VSV-G)). 2.2 pseudotypes were targeted with 1G8 antibody. The percentage of enhanced green fluorescent protein (EGFP) positive cells and mean fluorescence intensity are indicated in each plot. The gates were set using parallel mock infections. (b) Table indicating the specificity index of FPhLucW (VSV-G), FPhLucW (2.2) + 1G8, and FUhLucW (2.2) + 1G8. Specificity index is calculated as relative luciferase units (RLU)/μg protein obtained after transduction of LPSCA4 divided by the RLU/μg protein obtained in non-prostate cells. The non-prostate cells analyzed are indicated on the left in the table. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Dual-targeted lentiviral vectors target bone metastases of prostate cancer in vivo. (a) Analysis of 2.2 pseudotype background. Three naïve male severe combined immunodeficiency (SCID) mice were injected with FUcfLW (2.2) + isotype control antibody (15 μg/ml) and three others with FUcfLW (vesicular stomatitis virus glycoprotein (VSV-G)) through the tail vein. cfL is a flag-tagged version of humanized luciferase. The luciferase expression of two animals from each group on day 14 is shown. The maximum photon output for the 2.2 group was 8.8 × 104 and 3.4 × 104 p/s/cm2/sr and for the VSV-G group 8.4 × 106 and 6.5 × 106. (b) 1 × 105 LAPC-9 cells were implanted into the left tibia of male SCID mice. On days 7 and 14 after implantation, FPcfLW (2.2) with 1G8 or isotype control monoclonal antibody, or FPcfLW (VSV-G) was injected through the tail vein. The groups consisted of 16, 5, and 5 mice respectively. Five mice were injected with phosphate-buffered saline as controls. Mice were imaged in a cooled charged-coupled device (CCCD) camera and X-rayed once a week for 33 days. The images shown are from day 33 and the X-rays of the left leg from day 30. CCCD imaging of two representative animals from each group are shown. X-ray and organs of animal #2 are shown. Bone, bones of the lower leg (tibia and fibula); tumor, soft tumor that surrounds the bone. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Luciferase expression can be detected in the metastasis before X-ray manifestation. X-ray and cooled charged-coupled device (CCCD) images of one animal between 2 and 5 weeks post LAPC-9 cell implantation in the tibia. FPcfLW (2.2) + 1G8 antibody was injected on weeks 1 and 2 after LAPC-9 cell injection. An optical signal can be detected at 3 weeks after cell implantation (1 week after the second vector injection), when there is no pathology apparent on the X-ray. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Combined targeting achieves prostate cell-specific transgene expression. Tissue sections obtained from the left hind limbs of animals with LAPC-9 bone tumors were processed for immunohistochemistry. The vectors injected into the tail veins of the mice are indicated to the left of the panels. Luciferase expression, as detected by α-flag antibody, is shown in red; human cell nuclei are detected by α-hPARP antibody (green) and all nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) and shown in blue. Magnification is ×200. Brown bar represents 100 μm. Molecular Therapy 2007 15, 1973-1981DOI: (10.1038/sj.mt.6300271) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions