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An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice

Jun Yu^*, Li Lei, Yuxin Liang, Linda Hinh, Reed P. Hickey, Yan Huang, Dinggang Liu, Jennifer L. Yeh, Edward Rebar, Casey Case, Kaye Spratt, William C. Sessa^* and Frank J. Giordano^,1

^* Department of Pharmacology;
Department of Medicine, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Connecticut, USA; and
Sangamo Biosciences, Richmond, California, USA

¹Correspondence: BCMM 436C, 295 Congress Ave., New Haven, CT 06443, USA. E-mail: frank.giordano{at}yale.edu

SPECIFIC AIMS

Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. The current study was conducted to investigate the biological effects of an engineered VEGF-activating ZFP in a model of hindlimb ischemia in which spontaneous recovery does not occur and to compare the in vivo expression profiles of representative angiogenesis-associated genes in response to either ZFP-induced VEGF gene activation or to cDNA-based expression of the single VEGF164 splice variant.

PRINCIPAL FINDINGS

1. MVZ+426 (Fig. 1A, B ), an engineered zinc finger protein transcription factor designed to specifically activate expression of the endogenous vascular endothelial growth factor gene (VEGFa), induced expression of VEGFa in skeletal muscle in vivo to equivalent total protein levels as those induced by either VEGF164 cDNA or the hypoxia-inducible factor HIF-1 (Fig. 1C, D )

View larger version (13K): [in this window] [in a new window]   Figure 1. Adenovirus-mediated expression of an engineered VEGFa-activating ZFP promotes limb salvage in older C57B6 mice with surgically-induced limb ischemia. A) The amino acid sequence of each individual zinc finger is depicted above the corresponding base triplet to which it binds. B) The VEGF-activating ZFP expression cassette, as subcloned into the adenovirus vector, is schematically represented (CMV=cytomegalovirus promoter; NLS=nuclear localization sequence, p65TAD=the p65 transactivation domain). C) Real-time quantitative RT-PCR of total RNA from hindlimb skeletal muscle injected with Ad-MVZ+426b demonstrates increased VEGFa mRNA levels (expressed as fold increase relative to the contralateral limb injected with control adenovirus encoding ß-galactosidase; n=3/group). D) Western blot analysis demonstrates that MVZ+426b induces expression of VEGF protein at a level equivalent to VEGF expression induced by the hypoxia-inducible factor HIF-1 and by VEGF164 cDNA. E) MVZ+426b-mediated VEGF expression was associated with a significant improvement in limb salvage at wk 1, 2, and 4 postsurgical induction of hindlimb ischemia MVZ+426b (B); 0 = normal, 1 = pale foot or abnormal gait, 2 = gangrenous tissue < 50% of foot without lower limb muscle necrosis, 3 = gangrenous tissue < 50% of foot with lower limb muscle necrosis, 4 = gangrenous tissue > 50% of foot, 5 = loss of 50% lower limb. * 0.05, ** 0.005.

2. Adenovirus-mediated expression of the ZFP MVZ+426 in skeletal muscle resulted in significantly improved limb salvage and function in advanced-age mice with severe hindlimb ischemia (Fig. 1E )

3. MVZ+426-induced expression of endogenous VEGFa resulted in significantly improved blood flow to the ischemic hindlimb in advanced-age mice, evident at 1 wk post-induced ischemia and sustained at 4 wk (Fig. 2A )

View larger version (55K): [in this window] [in a new window]   Figure 2. ZFP-induced VEGF expression increases blood flow and vessel counts in ischemic hindlimbs. One wk after MVZ+426b gene delivery there was a significant increase in the ischemic limb/contralateral limb blood flow ratio as compared with Ad-RFP treated control mice. This improvement remained significant 2 and 4 wk after MVZ+426b gene delivery (A). Analysis of vessel density by anti-Pecam immunostaining demonstrated increased vessel counts in the MVZ+426b-transduced muscles (B, C); n = 5/group; * 0.05; ** 0.005.

This increased flow was accompanied by significantly greater vessel counts in the ischemic hindlimb (Fig. 2B, C ).

4. ZFP-induced expression of the endogenous VEGFa gene with consequent expression of all naturally occurring VEGFa splice variants, results in a significantly different expression profile of downstream angiogenesis-associated genes than does expression of the single splice variant VEGF164 (murine form of VEGF165, which has been used in clinical trials)

These differences included asymmetrically increased expression of several angiogenic growth factors (e.g., acidic fibroblast growth factor, midkine, insulin-like growth factor 1, platelet-derived growth factor B, placental growth factor, and TGFß3), angiogenesis-associated receptors and adhesion complexes (e.g., VCAM-1, PECAM, ephrin B4, integrin b3, TGF receptor 1, and PDGF receptors and ß), and a variety of other angiogenesis-associated genes (e.g., osteopontin, thrombospondins 2 and 3, interleukin 10, Smad1, and others; see Table 1, complete online version).

CONCLUSIONS AND SIGNIFICANCE

Here we show that an engineered zinc finger protein designed to activate the endogenous VEGFa gene is able to ameliorate hindlimb ischemia and promote limb salvage in older C57BL6 mice. We recently established that VEGF-activating ZFPs could induce angiogenesis and promote wound healing in vivo, and now demonstrate the ability of these engineered transcription factors to ameliorate chronic ischemia in a clinically applicable model. C57BL6 mice 6 months of age demonstrate an attenuated endogenous recovery from induced hindlimb ischemia relative to younger mice, and the efficacy of MVZ+426b in this model is of relevance to the older peripheral vascular disease patient population. The present study constitutes further documentation that engineered ZFPs are effective in vivo and can be used to modulate biological responses in a therapeutic manner. These data also demonstrate that there is indeed a biological role for splice variants and that simple cDNA-based expression of a single splice variant may not result in an equivalent biological effect as is induced by endogenous gene activation.

The ability to design molecules de novo to regulate the transcriptional expression of specifically targeted genes has significant clinical potential and represents a new direction in the field of gene therapy. This approach may have definitive advantages over standard gene therapy approaches, including the ability to target several genes as once and the capacity to induce all the expected splice variants of a specific gene.

View larger version (22K): [in this window] [in a new window]   Figure 3. Engineering zinc finger protein transcription factors. ZFP design is facilitated by understanding the chromatin structure of the gene targeted. One approach is DNase hypersensitivity mapping, which defines open chromatin regions that will allow accessibility of the engineered ZFP (A). Next, current knowledge of the relationship between ZFP protein structure and DNA binding specificity is used to construct ZFP fingers to bind a specific DNA sequence in the regulatory region of the targeted gene (B). Each finger binds a base pair triplet on the sense strand, thus by combining several fingers DNA sequences of variable length can be targeted (e.g., the 3-finger ZFP depicted in panel B binds a 9 base pair sequence). Next, the DNA binding ZFP domain is fused to an activation domain to make a functional ZFP (C); TAD = transactivation domain. For ZFP transcription factors this can be either a transcription repressor or activator. For gene correction, the ZFP can be fused to an endonuclease domain, thus directing site-specific DNA cutting.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3670fje;

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This Article Full Text (PDF) All Versions of this Article: 20/3/479 04-3670fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yu, J. Articles by Giordano, F. J. Search for Related Content PubMed PubMed Citation Articles by Yu, J. Articles by Giordano, F. J.