Volume 12, Issue 1, Pages 49-57 (July 2005) Noninvasive Imaging of ex Vivo Intracoronarily Delivered Nonviral Therapeutic Transgene Expression in Heart  Luyi Sen, Sanjiv S. Gambhir, Hiroshi Furukawa, David B. Stout, Allison Linh Lam, Hillel Laks, Guanggen Cui  Molecular Therapy  Volume 12, Issue 1, Pages 49-57 (July 2005) DOI: 10.1016/j.ymthe.2005.03.004 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Efficiency of ex vivo intracoronarily delivered and liposome-mediated reporter–therapeutic linked gene transfection in cardiac allografts. (A) Construction of the plasmid vector containing both the PET reporter gene HSV1-sr39tk and the therapeutic gene human IL-10, which are driven by two identical CMV promoters. (B) The efficiency of gene transfection was compared among the donor hearts transfected with reporter gene pCMVsr39tk alone (n = 8), therapeutic gene pCMVhIL-10 alone (n = 8), or reporter–therapeutic linked gene (pCMVsr39tk-CMVhIL-10, n = 8) at p.o.d. 8. (C) (Top) Representative data from quantitative RT-PCR analysis of sr39tk and hIL-10 transgene expression in LV of cardiac allografts (lanes 1 and 2) and recipient heart, lung, brain, liver, kidney, and skeletal muscle (lanes 3–8). (Bottom) Quantitative RT-PCR analysis shows the overexpressed reporter and therapeutic transgene homogeneously distributed in LV, IVS, RV, LA, and RA of a cardiac allograft. (D) Dose dependence of sr39tk and hIL-10 transgene expression in cardiac allografts summarized with a histogram. Cardiac tissue samples were collected on p.o.d. 8. Quantitative sr39tk and hIL-10 transgene cDNA expression levels were plotted as a ratio to the expression of the housekeeping gene GAPDH (*P < 0.05). (E) Time dependence of sr39tk and hIL-10 gene expression in allografts treated with reporter–therapeutic linked gene versus “empty” liposome assessed by RT-PCR. To determine the time course of the transgene and protein expression, in the Lip-sr39TKhIL-10-treated group animals were sacrificed and left ventricular tissue was collected for analysis at p.o.d. 0 (n = 5), 2 (n = 5), 4 (n = 5), 6 (n = 8), 8 (n = 15), 12 (n = 5), 18 (n = 5), or 28 (n = 5). In the control group treated with empty liposome, most allografts could survive for only 8 days; therefore animals were sacrificed and left ventricular tissue was collected for analysis at p.o.d. 0 (n = 5), 2 (n = 5), 4 (n = 5), 6 (n = 8), or 8 (n = 15). Molecular Therapy 2005 12, 49-57DOI: (10.1016/j.ymthe.2005.03.004) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Reporter–therapeutic gene transfection induced HSV1-sr39tk and hIL-10 protein overexpression. (A) Time dependence of TK and IL-10 protein overexpression in cardiac allografts. (B) Immunofluorescence staining shows the colocalization of TK and IL-10 protein expression in the cardiac allografts. (C) Representative result of Western blot analysis shows the homogeneous distribution of overexpressed TK and IL-10 protein in the LV, RV, IVS, LA, and RA. (D) Correlation between TK and IL-10 protein levels in cardiac allografts transfected with reporter–therapeutic linked gene. Molecular Therapy 2005 12, 49-57DOI: (10.1016/j.ymthe.2005.03.004) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Micro-PET imaging of[18F]FHBG accumulation in the myocardium of rabbit cardiac allografts. (A) Representative transaxial images of a donor rabbit cardiac allograft implanted in the neck of a recipient rabbit that was intracoronarily delivered ex vivo with liposome–pCMVsr39tk-CMVhIL-10. At p.o.d. 4, the distinct tracer accumulation was seen. At p.o.d. 8 a homogeneous distribution of[18F]FHBG accumulation in the myocardium of LV was observed. A decline of[18F]FHBG activity was seen at p.o.d. 18 and 28, while LV thickening occurred due to the acute allograft rejection. In contrast,[18F]FHBG accumulation was not observed in allografts treated with empty liposome or liposome–pCMVhIL-10 at p.o.d. 8. (B) Time course of %ID for myocardial[18F]FHBG accumulation calculated from micro-PET images serially scanned in 15 rabbits. (C) Micro-PET imaging of localization of reporter–therapeutic linked transgene/[18F]FHBG probe accumulation in comparison with the metabolic probe,[18F]FDG, accumulation. Molecular Therapy 2005 12, 49-57DOI: (10.1016/j.ymthe.2005.03.004) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Tomographic view of whole heart micro-PET image. (A)[18F]FHBG images demonstrating the homogeneously distributed reporter–therapeutic gene expression in the whole heart and[18F]FDG images showing the viable myocardium. Color scale is expressed as %ID/g. (B) Correlation between[18F]FHBG accumulation (%ID/g) and ex vivo γ counting of explanted heart or Western blot quantification of TK protein expression level in the myocardium tissue. (C) Correlation between IL-10 gene expression and[18F]FHBG/[18F]FDG ratio or[18F]FHBG accumulation (%ID/g) in the donor hearts. Molecular Therapy 2005 12, 49-57DOI: (10.1016/j.ymthe.2005.03.004) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Effects of reporter–therapeutic linked gene transfection in cardiac allografts on cardiac function and the efficacy of gene therapy. (A) Comparison of mean survival in cardiac allografts treated with empty liposome (Lip; n = 15), liposome–pCMVsr39tk (Lip-TK; n = 15), liposome–pCMVhIL-10 (Lip-IL-10; n = 15), or liposome–pCMVsr39tk-CMVhIL-10 (Lip-TK-IL-10; n = 15). (B) Representative histologic findings (H&E staining) in left ventricular tissue of cardiac allografts treated with empty liposome (Lip), liposome–pCMVsr39tk (Lip-TK), liposome–pCMVhIL-10 (Lip-IL10), or liposome–pCMVsr39tk-CMVhIL-10 (Lip-TK-IL10) at p.o.d. 8. (C) Comparison of the rejection score in the cardiac allografts affected by the empty liposome (Lip; n = 15), liposome–pCMVsr39tk (Lip-TK; n = 15), liposome–pCMVhIL-10 (Lip-IL10, n = 15), or liposome–pCMVsr39tk-CMVhIL-10 (Lip-TK-IL10; n = 15). (D) Comparison of the CD3+ lymphocyte infiltration in the cardiac allografts reduced by liposome–pCMVhIL-10 gene therapy and reporter–therapeutic linked gene therapy. (E) Comparison of LV systolic pressure in cardiac allografts treated with empty liposome (Lip; n = 15), liposome–therapeutic gene only (Lip-IL-10; n = 15), or reporter–therapeutic linked gene therapy (Lip-TK-IL-10; n = 15) to that in cardiac isografts (n = 15, *P < 0.05) and allografts without any treatment (n = 15, **P < 0.05). Systolic pressure was recorded at p.o.d. 4. (F) Incidence of arrhythmia that includes supraventricular tachycardia, atrial flutter and fibrillation, and ventricular tachycardia and fibrillation in the cardiac allografts treated with empty liposome (Lip; n = 15), liposome–therapeutic gene only (Lip-IL10; n = 15), reporter–therapeutic linked gene therapy (Lip-TK-IL10; n = 15), cardiac isografts (n = 15), and allografts without any treatment (n = 15) at 24 h after commencement of reperfusion. ECG was recorded continuously for 1 h. Values are expressed as a percentage of total cases. Molecular Therapy 2005 12, 49-57DOI: (10.1016/j.ymthe.2005.03.004) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions