Efficient Vascular Endothelial Gene Transfer Following Intravenous Adenovirus Delivery (2008)

Efficient intravascular gene delivery to the endothelium in defined vascular beds pertinent to pathological states has been a long-pursued goal for many gene therapy applications. Clear advantages include the minimally invasive route of delivery and the ability to target distal sites in the body that are relatively inaccessible surgically. Such targets are diverse and include angiogenic endothelium in cancer, the vasculature of defined organs such as the lung, and microvascular endothelium in regions of poor blood flow. Many research endeavors have investigated strategies for achieving endothelium-targeted gene delivery by the intravascular route. A major obstacle that has limited development is the clear propensity for liver targeting by many gene therapy vectors, thus limiting the availability for vector uptake at other sites. Despite this, a small number of reported successes in vascular targeting by the intravascular route have been achieved via transductional targeting (tropism modification) of the vector in the case of adenovirus1Reynolds PN Nicklin SA Kaliberova L Boatman BG Grizzle WE Balyasnikova I et al.Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.Nat Biotechnol. 2001; 19: 838-842Crossref PubMed Scopus (213) Google Scholar and defined engineering of nonviral vectors.2Hood JD Bednarski M Frausto R Guccione G Reisfeld RA Xiang R et al.Tumor regression by targeted gene delivery to the neovasculature.Science. 2002; 296: 2404-2407Crossref PubMed Scopus (793) Google Scholar In this issue of Molecular Therapy, Tal et al.3Tal R Shaish A Rofe K Feige E Varda-Bloom N Afek A et al.Endothelial-targeted gene transfer of hypoxia-inducible factor-1α augments ischemic neovascularization following systemic administration. 2008; (Mol Ther 16: 1927–1936.)Google Scholar report successful vascular endothelial cell transduction following intravascular adenovirus administration. The authors engineered first-generation recombinant adenoviral vectors to express a constitutively active version of hypoxia-inducible factor-1α (mHIF1α) from either the cytomegalovirus immediate early promoter (CMV IEP) or a modified version of the murine pre-proendothelin promoter (PPEx3). PPEx3 contains three copies of the endothelial cell (EC)-specific regulatory region found in the endogenous pre-proendothelin gene4Bu X Quertermous T Identification of an endothelial cell-specific regulatory region in the murine endothelin-1 gene.J Biol Chem. 1997; 272: 32613-32622Crossref PubMed Scopus (23) Google Scholar and has been reported to drive efficient transgene expression in tumors following intravascular delivery of adenoviral vectors.5Greenberger S Shaish A Varda-Bloom N Levanon K Breitbart E Goldberg I et al.Transcription-controlled gene therapy against tumor angiogenesis.J Clin Invest. 2004; 113: 1017-1024Crossref PubMed Scopus (59) Google Scholar In the current study, Tal et al. demonstrate that PPEx3-driven mHIF1α expression stimulates angiogenesis in in vitro EC tube formation assays at twice the efficiency of the CMV IEP. Next, the authors assessed CMV IEP- or PPEx3-driven green fluorescent protein (GFP) expression in a mouse model of hindlimb ischemia (induced by femoral artery ligation) after intravascular administration of each virus. Interestingly, whereas nonspecific expression of GFP was detected in both ischemic and nonischemic skeletal muscle fibers when GFP was driven by the CMV IEP, PPEx3-driven GFP expression was observed only in ECs of the vasculature within ischemic muscle. When used to express the therapeutic transgene, the majority of CMV IEP-driven expression was observed in the liver. Conversely, PPEx3-mediated mHIF1α expression was negligible in the liver concomitant with an efficient level of expression detected selectively in the endothelium of the target ischemic muscle. This resulted in a significant increase in capillary density and restored blood flow to approximately 45% of that observed for noninjured animals; it also significantly reduced limb necrosis. In stark contrast, intravascular administration of adenoviruses encoding mHIF1α from the CMV IEP resulted in no therapeutic benefit but did induce liver toxicity, as indicated by significant increases in serum bilirubin, aspartate aminotransferase, and alanine aminotransferase levels. It was surprising that there was no observable liver toxicity when the PPEx3 promoter was used to drive mHIF1α expression, because Kupffer cell uptake should be identical for both viruses. This study yielded somewhat unexpected findings. First and foremost, the authors achieved highly efficient and selective vascular targeting of gene expression by this route. Most previous work has focused on removing native adenoviral tropism for the coxsackie and adenovirus receptor (mediated via the fiber) and/or the integrin interaction (mediated by penton base) and providing new vascular-selective tropism at the transductional level. These studies provided conflicting results, with many groups not showing any significant redistribution of transgene expression away from the liver and to the vasculature (for review, see ref. 6Nicklin SA Wu E Nemerow GR Baker AH The influence of adenovirus fiber structure and function on vector development for gene therapy.Mol Ther. 2005; 12: 384-393Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The former inability to remove native adenoviral tropism for the liver is now known to be due to the interaction of host coagulation factor X with the adenoviral hexon protein.7Lowenstein PR With a little help from my f(X)riends!: the basis of Ad5-mediated transduction of the liver revealed.Mol Ther. 2008; 16: 104-106Google Scholar The most elegant finding of the study by Tal et al.3Tal R Shaish A Rofe K Feige E Varda-Bloom N Afek A et al.Endothelial-targeted gene transfer of hypoxia-inducible factor-1α augments ischemic neovascularization following systemic administration. 2008; (Mol Ther 16: 1927–1936.)Google Scholar is that therapeutic targeting is seemingly achieved through the use of transcriptional targeting alone. The efficiency and selectivity of the delivery seem to match those of the previous benchmark, dual transductional and transcriptional targeting of adenoviral vectors to pulmonary endothelium.1Reynolds PN Nicklin SA Kaliberova L Boatman BG Grizzle WE Balyasnikova I et al.Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.Nat Biotechnol. 2001; 19: 838-842Crossref PubMed Scopus (213) Google Scholar This highlights a key, hitherto-unrecognized issue that identification of a highly efficient endothelium-regulatory element to drive transgene expression is sufficient to achieve therapeutic benefit by the intravascular route. This study also demonstrates that ectopic expression of transgenes from the CMV IEP may play a significant role in induction of liver toxicity at later time points (for example, 5 days). Although this obviously could be transgene-specific (e.g., with the use of cytotoxic transgenes), it is notable that PPEx3-driven expression of mHIF1α induces less liver toxicity than does CMV IEP-driven expression of either mHIF1α or luciferase. In summary, this study should provide new impetus for the identification of efficient gene-regulatory elements that impart efficacy when engineered into adenoviral vector—creating further impetus to develop minimally invasive and targeted therapeutic agents. These findings will be met with interest by researchers in the cardiovascular disease and cancer gene therapy fields and might guide development of the next generation of targeted gene therapeutics.