1. Volume 3, Issue 4, Pages 507-515 (April 2001)
Two Animal Models of Retinal Degeneration Are Rescued by Recombinant Adeno- associated Virus-Mediated Production of FGF-5 and FGF-18 
Eric S. Green, Katherine G. Rendahl, Shangzhen Zhou, Martha Ladner, Mazie Coyne, Ranjana Srivastava, William C. Manning, John G. Flannery 
Molecular Therapy 
Volume 3, Issue 4, Pages (April 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
The pD10-CMV-FGF-5 and pD10-CMV-FGF-18 vector constructs contain an expression cassette including a stuffer sequence (PhiX174), a CMV immediate/early promoter plus enhancer (CMV IE ENH/PRO), and a bovine growth hormone polyadenylation site (BGH pA), flanked by inverted terminal repeat sequences (ITR). The FGF-5 vector is shown here. cDNA sequences are from human FGF-5 and FGF-18.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Injections of all three types of virus on P15 produced extensive expression of the protein driven by the CMV promoter. The cellular localization of each of the expressed proteins, 3 weeks postinjection, is shown here. Control AAV-LacZ-injected eyes (A) show some protein expression in the outer nuclear layer, but most expression is in the inner and outer segments of photoreceptors. A small number of ganglion cells and RPE cells also express LacZ (not shown). In AAV-FGF-5- (B) and AAV-FGF-18- (C) injected eyes, expression was restricted to the inner and outer segments. Labeling is not seen in uninjected eyes (D). Scale bar, 15 μm. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Examples of an AAV-FGF-5-injected eye (A), an AAV-FGF-18-injected eye (B), an AAV-LacZ-injected eye (C), and an uninjected eye (D). All eyes were from S334ter-4 animals, and all photographs were taken from along the midline in the inferior hemisphere. Scale bar, 15 μm. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Outer nuclear layer thickness measurements 45 days postinjection. AAV-FGF-5 and AAV-FGF-18 injections rescued photoreceptors in both the inferior (A) and the superior (B) hemispheres in S334ter-4 rat retinas. The numbers of eyes used in the S334ter-4 experiments were 15 (FGF-5), 10 (FGF-18), 14 (LacZ), 6 (PBS), 8 (uninjected), and 3 (Wt). In P23H-3 rats, there was near complete rescue from cell death in both hemispheres (C and D). The numbers of eyes used in the P23H-3 experiments were 5 (FGF-5), 7 (LacZ, PBS, and uninjected), and 3 (Wt). FGF-5, AAV-FGF-5-injected eyes; FGF-18, AAV-FGF-18-injected eyes; LacZ, AAV-LacZ-injected eyes; PBS, PBS-injected eyes; Uninj, uninjected eyes; Wt, uninjected wild-type eyes.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Amplitudes of ERG A waves and B waves in injected and uninjected S334ter-4 rat eyes. (A) There were no significant differences in A-wave amplitudes among all sets of eyes. (B) B waves of uninjected eyes were significantly greater than those of all treatments except PBS injections. Error bars show the standard deviation among averaged amplitudes from 6 to 10 eyes. FGF-5, AAV-FGF-5-injected eyes; FGF-18, AAV-FGF-18-injected eyes; LacZ, AAV-LacZ-injected eyes; PBS, PBS-injected eyes; Uninj, uninjected eyes.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
In regions expressing growth factors after viral injection, shown for FGF-18 in (A), we found elevated levels of expression of the growth factor receptors FGFR1 and FGFR2 (B and C). Both receptors localized to the photoreceptor inner and outer segments, just as did the growth factors themselves. No labeling was found in regions distant from the injection site or when primary antibody was omitted from the staining protocol (D and E). Sections from throughout the retinas of two AAV-LacZ-injected eyes were also labeled as controls and showed nostaining with either anti-FGFR1 (F) or anti-FGFR2 antibodies. All animals are wild-type. Scale bar, 15 μm. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Transient transfections were performed on 293 cells in tissue culture to examine the secretion patterns of the FGF-5 and FGF-18 proteins produced by our vectors. (A) After transfection with the plasmid pD10-CMV-FGF-5, protein was detected only in the cell lysate. Lane 1 represents 50 ng of 29.5-kDa recombinant FGF-5 protein. Lanes 2 and 3, showing FGF-5 expression, are cell lysates from cells transfected with two different clones of the plasmid. Lane 4 is lysate from cellstransfected with a negative control plasmid, CMV-Epo. Lanes 5 and 6 represent media from cells transfected with the two plasmid clones. Lane 7 is medium from the CMV-Epo plasmid. (B) After transfection with the plasmid pD10-CMV-FGF-18, protein was detected primarily in the cell lysate, but to some degree also in the media. Lanes 1–3 represent 1, 2, and 10 ml of tissue culture medium from cells transfected with the pD10-CMV-FGF-18 plasmid. Lane 4 is blank. Lanes 5, 6, and 7 contain 2, 10, and 20 ml of lysate from the transfected cells. Lanes 8 and 9 are negative controls which contained 20 ml of tissue culture medium and cell lysate from uninfected cells, respectively. Lane 10 contains a positive control of FGF-18-maltose binding protein fusion, predicted to be larger (80 kDa) than the FGF-18 protein (about 40 kDa). The separate bands for FGF-18 are likely due to N-linked glycosylation.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions