The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting

Abstract

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP)1.This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biologic production in previously inaccessible organs that initiate the most debilitating or lethal human diseases.

Pulmonary vasculature directed adenovirus increases epithelial lining fluid alpha-1 antitrypsin levels

“…To confirm that the re‐targeted Ad5.MBP.CMV.A1AT retains the lung targeting gains demonstrated previously , we systemically injected female C57BL/6 J mice between the ages of 7–10 weeks with 10 10 viral particles of either Ad5.MBP.CMV.A1AT or Ad5.CMV.A1AT via the tail vein. The mice were then sacrificed at 8, 24 or 72 h and the lungs, livers and spleens were collected to determine the biodistribution of the viruses by DNA PCR amplification ( n = 5 for each virus at each time point).…”

“…Briefly, pShuttleCMV.A1AT was linearized with Pme I and recombined with pAdEasy‐1 in BJ5183 and selected for in a kanamycin infused agar plate to generate pAd.CMV.A1AT. In parallel, to generate the MBP version of the same plasmid, the positive clone of pShuttleCMV.A1AT was linearized with Pme I and recombined with the pAd.MBP.CMV.Luc that we have described previously in E. coli strain BJ5183 . The recombinant adenoviral plasmids were linearized by PacI digested and transfected into 293‐F28 cells using Superfect reagent (Qiagen, Valencia , CA, USA ).…”

“…In this regard, we have recently developed strategies to retarget adenoviral serotype 5 vectors (Ad5) that allow selective in vivo gene delivery. A recent approach employs the incorporation of a myeloid binding peptide (MBP) into the Ad5 fiber knob, achieving two‐fold targeting goals: mitigation of the native Ad5 hepatotropism and dramatically enhanced gene delivery to the pulmonary endothelium .…”

A New Gorilla Adenoviral Vector with Natural Lung Tropism Avoids Liver Toxicity and Is Amenable to Capsid Engineering and Vector Retargeting

“…The GFP reporter fluorescence in lung rose rapidly after virus injection reaching a peak level by day 1 and remained high by days 3, 5, and 7 ( Figures 4A, lung Figure 4B). Of note, the lung EC gene transfer efficiency by GAd (52% GFP + ) at day 3 was at least 2.4-fold of what was attained by the EC-targeted Ad5 with myeloidbinding peptide (MBP)-incorporated capsid fiber in a side-by-side comparison experiment ( Figure 4C; HAd5.MBP; 21% GFP + ECs) (6). The GAd exhibited a shorter duration of vector expression in spleen for up to 5 days post virus injection, and the expression was reduced by day 7 (Figure 4A, spleen).…”

Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection

“…As a consequence, the ability to permanently modify endothelial cells through gene augmentation represents an exciting therapeutic avenue for the treatment of chronic vascular disease, potentially allowing long-term treatment to be achieved after a single intervention. However, previous efforts to target vascular endothelial cells in vivo through intravenous administration of native DNA 17 or of recombinant adeno-associated viral (rAAV) 18,19 or adenoviral vectors 20,21 have proven to be ineffectual, resulting in minimal vascular transduction and, frequently, acute systemic toxicity. 22,23 Attempts have been made to improve on the native properties of rAAV vectors by introducing targeted mutations within the shared C-terminal domain of the structural proteins (VP1/VP2/VP3) that comprise the viral capsid (see Fig.…”