1. Volume 12, Issue 4, Pages 716-724 (October 2005)
Alteration of splicing signals in a genomic/cDNA hybrid VEGF gene to modify the ratio of expressed VEGF isoforms enhances safety of angiogenic gene therapy 
Hideki Amano, Neil R. Hackett, Robert J. Kaner, Paul Whitlock, Todd K. Rosengart, Ronald G. Crystal 
Molecular Therapy 
Volume 12, Issue 4, Pages (October 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Design of VEGF-All and VEGF-All6A+. VEGF-All is a human-derived cDNA/genomic hybrid consisting of the cDNA from exon 1 to exon 5 and the genomic configuration of exons 5–8 with the exception of an internal deletion to reduce the size of intron 7 from 2425 to 721 bp [27]. Alternative splicing, as indicated above VEGF-All, provides mRNA encoding the three major isoforms of VEGF: VEGF121 (exons 1–5 and 8), VEGF165 (exons 1–5, 7,and 8), and VEGF189 (exons 1–5, 6A, 7, and 8). VEGF-All6A+ was created by site-directed mutagenesis in which the native splicing sequences around exon 6A, also present in VEGF-All, were enhanced to resemble the consensus. The splice acceptor and branch site upstream of exon 6A and the splice donor downstream of exon 6A were modified as shown in the lowercase bold font to favor inclusion of exon 6A in the mRNA, thus promoting a higher yield of VEGF189 and less VEGF121 and VEGF165.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
VEGF expression patterns in cells infected by AdVEGF-All and AdVEGF-All6A+. Rat2 cells were infected with AdVEGF-All and AdVEGF-All6A+ (103 particle units/cell) and after 24 h transferred to serum-free medium. After a further 24 h, the media and cells were collected separately. (A) VEGF secreted into media assessed by ELISA after collection of media with (+) or without (−) the prior addition of 5 units/ml heparin. (B) Relative levels of VEGF isoform-specific mRNAs. Total RNA was extracted from the cells and converted to cDNA. Real-time PCR was performed with TaqMan primers and probes specific to the three VEGF isoforms as indicated below the axis. The amount of each mRNA was calculated relative to the rRNA content for the same sample also determined by TaqMan. Data are presented as means ± standard deviation of triplicates.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
VEGF expression in mouse hind-limb muscle. C57Bl/6 mice (n = 5/group) were administered 1010 particle units of AdVEGF-All or AdVEGF-All6A+ by injection into the quadriceps. After 2 days the mice were sacrificed and the quadriceps was harvested. Total RNA was extracted and converted to cDNA. (A) The cDNA for one mouse per group was assessed by RT-PCR using primers that give different-sized products for each isoform. The expected location of the PCR products for each isoform are indicated by the arrows. (B) VEGF mRNA isoforms assessed by real-time PCR with TaqMan primers and probes specific to the VEGF isoforms shown below the axis. The relative amounts of the VEGF mRNA isoforms are expressed relative to the level of rRNA. The mean ratio for the AdVEGF-All group was used for normalization for each isoform. Each data point represents a single mouse.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Recovery from hind-limb ischemia mediated by AdVEGF-All and AdVEGF-All6A+ vectors. C57Bl/6 mice (n = 5 per group) were administered vector to the quadriceps at various doses immediately after excision of a section of the external iliac artery. The blood flow in the ischemic limb relative to that in the control limb was assessed presurgery, immediately postsurgery, and at intervals afterward by laser Doppler scanning. (A) Time course of recovery using a vector dose of 105 particle units. (B) Dose dependence of recovery at 4 weeks postvector. Data are plotted as means ± standard deviation.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Histologic assessment of angiogenesis mediated by AdVEGF-All and AdVEGF-All6A+. Quadriceps muscle was isolated from mice 28 days after injection with 105 pu of AdNull, AdVEGF-All, or AdVEGF-All6A+ and endothelial expression of PECAM-1 was assessed by immunohistochemistry. (A) Histology, AdNull. (B) Histology, AdVEGF-All. (C) Histology, AdVEGF-All6A+. Scale bar = 100 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Mortality following intravenous administration of high-dose AdVEGF-All and AdVEGF-All6A+ vectors. C57Bl/6 mice (n = 6/group) were injected intravenously with 5 × 109 or 5 × 1010 pu of AdNull, AdVEGF-All, and AdVEGF-All6A+. (A) Mortality following intravenous administration of 5 × 109 pu of the vectors. (B) Mortality following intravenous administration of 5 × 1010 pu of the same vectors.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Tumor growth after intravenous administration of the AdVEGF-All and AdVEGF-All6A+ vectors. Lewis lung carcinoma cells (3 × 105 cells in 100 μl) were injected subcutaneously into C57Bl/6 mice (n = 8 each cohort). On day 3, when the tumor volume was approximately 62.5 mm3, Ad vectors (108 pu) were injected intravenously and volume was assessed in the same mice at 3-day intervals. Data are presented as means ± standard deviation.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Pulmonary edema following intratracheal injection of VEGF vectors. C57Bl/6 mice (n = 7 per group) were administered 5 × 1010 particle units of each vector by the intratracheal route. (A–C) Morphology. Lungs were collected for pathological examination after 5 days. (A) AdNull. (B) AdVEGF-All. (C) AdVEGF-All6A+. Hematoxylin and eosin staining. Bar, 100 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions