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). 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 the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the 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 biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Figure 1. Incorporation of MBP into Ad5 drastically increased viral gene expression to vascular beds of multiple host organs

(A) Immunofluorescence microscopy analysis of vector EGFP expression in host organs following intravenous injection of 1×10¹¹ viral particles (vp) of Ad.MBP.CMV into adult C57BL/6J mice revealed prominent transgene expression in lung, heart, kidney, gastrocnemius muscle, pancreas, small and large bowel, and brain. Co-staining of tissue sections with an EC-specific endomucin/CD31 cocktail revealed that EGFP expression was restricted to the vasculature. (B) EGFP fluorescence per µm² of tissue section area (FI, fluorescence intensity) in each organ derived from Ad5.CMV-injected mice (n=4 for all organs) versus that from Ad.MBP.CMV-injected mice (n=10 for liver, spleen, heart, kidney, muscle, small bowel, and brain; n=7 for lung, pancreas, and large bowel). (C) The percentage of vascular EC area expressing EGFP in each organ derived from Ad5.CMV-injected mice (n=4 for all organs) versus that from Ad.MBP.CMV-injected mice (n=10 for heart, kidney, muscle, small bowel, and brain; n=7 for lung, pancreas, and large bowel). Bar graph is mean +/− standard deviation asterisk: adjusted p<0.05. Magnification: 100×, Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI. Li: liver, S: spleen, Lu: lung, H: heart, K: kidney, M: muscle, P: pancreas, SB: small bowel, LB: large bowel, B: brain.

Figure 2. Warfarin pretreatment reduced Ad.MBP.CMV liver tropism but did not alter gene expression in other host organs

(A) Warfarin, 5 mg/kg, on day −3 and −1 before vector injection diminished hepatocyte expression but did not change transgene expression in spleen. (B) EGFP fluorescence per µm² of tissue area in each organ derived from warfarin-treated mice (n=3 for all organs) normalized as percentage of the mean value of vehicle-treated or untreated counterparts (n=10 for liver, spleen, heart, kidney, muscle, small bowel, and brain; n=7 for lung) with standard deviation. Warfarin pretreatment reduced vector liver expression by 68% (Li) but did not lead to a significant change in gene expression in spleen (S), lung (Lu), heart (H), kidney (K), muscle (M), small bowel (SB), or brain (B). Asterisk indicates adjusted p<0.05. Magnification: 100×, Red: CD31/endomucin, Green: EGFP immunofluorescence, Blue: DAPI.

Figure 3. Systemic administration of a low dose of Ad.MBP.CMV into adult mice produced differential and non-linear reduction in gene expression in host organs

(A) EGFP expression in host liver, spleen, lung, and brain following intravenous injection of 1×10¹¹ or 2×10¹⁰ vp of Ad.MBP.CMV into adult mice. Lowering vector dosage significantly reduced EGFP expression in vascular ECs of liver, spleen, and brain but did not change the expression in lung. (B) EGFP fluorescence per µm² of tissue area in each organ derived from the low-dose group (n=6 for each organ). (C) Normalization of the tissue EGFP fluorescence intensity values in (B) to the mean value of the high-dose counterparts. The spleen and brain EGFP expression in low-dose group was 16% and 31% of the high-dose counterparts. However, low virus dose drastically diminished the transgene expression in, heart, kidney, muscle, pancreas, and small bowel (3% of high-dose level). The low dose did not significantly alter the transgene expression in lung (91% of high-dose level). Asterisk indicates p<0.05. Magnification: 100×, Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI. Li: liver, S: spleen, Lu: lung, H: heart, K: kidney, M: muscle, P: pancreas, SB: small bowel, B: brain.

Figure 4. Depletion of circulating monocytes and hepatic and splenic macrophages lead to an increased Ad.MBP.CMV gene expression in the lung without a significant change in gene expression in other organs

(A) Representative flow cytometry plots (left panel) quantifying the FSC-high/SSC-low/CD11b-positive/CD45-positive monocyte population in circulation. Relative frequency (right panel) of circulating monocytes from clodronate liposome-treated mice (clod, n=3) versus saline-treated mice (veh, n=3). Intravenous injection of clodronate liposomes depleted circulating CD11b-positive cells by 84%. (B) EGFP fluorescence per µm² of tissue area in each organ derived from the saline-injected mice (n= 7 for liver, spleen, heart, kidney, muscle, pancreas, small bowel, and brain; n=4 for lung) versus clodronate-liposome-injected mice (n=8 for liver, spleen, heart, kidney, pancreas, small bowel, and brain; n=7 for muscle; n= 5 for lung). Intravenous clodronate increased Ad.MBP.CMV lung expression by 2-fold (Lu) but did not result in a significant change in gene expression in liver (Li), spleen (S), heart (H), kidney (K), muscle (M), pancreas (P), small bowel (SB), or brain (B). Asterisk indicates adjusted p<0.05.

Figure 5. Ad.MBP.ROBO4 detargeted hepatocyte expression but reduced levels of vascular EC expression in other host organs

(A) EGFP expression following intravenous injection of 1×10¹¹vp of Ad.MBP. ROBO4 into adult mice. Ad.MBP.ROBO4 yielded punctate vascular EC expression in liver but showed a reduced targeting efficiency to vascular ECs in spleen, lung, heart, kidney, muscle, small bowel, and brain. (B) The EGFP-positive vascular area analysis was performed as shown in Figure 1C. Magnification: 100×,Red: endomucin/CD31, Green: EGFP immunofluorescence, Blue: DAPI. Li: liver, S: spleen, Lu: lung, H: heart, K: kidney, M: muscle, SB: small bowel, LB: large bowel, B: brain.