Intravascular Delivery Research Articles

SC-24 * IMPROVEMENT OF HUMAN MESENCHYMAL STEM CELL HOMING IN DELIVERY OF THERAPEUTIC AGENTS TO GLIOMA STEM CELL XENOGRAFTS

There is an urgent need to develop novel delivery methods for brain tumors. To this end, we have developed human bone marrow mesenchymal stem cells (MSCs) to overcome the problems associated with traditional therapeutic treatments. Compared with other cellular delivery systems, MSCs may be easily acquired from patients, can be readily expanded and engineered ex vivo, can be auto-transfused without fear of rejection, and are free of ethical concerns. Therefore, MSCs are the ideal cells for therapeutic clinical applications. Defining the process of homing of MSCs to tumors-which is largely unknown-is essential to improve their application in clinical medicine. We have recently exploited glioma stem cell (GSC) xenografts (GSCXs) as a pertinent clinical model of human gliomas, because unlike GBM cell lines, GSCXs recapitulate the histology of the tumor from which they are derived. After intravascular delivery some GSCXs attract MSCs, while others do not. We hypothesized that successful homing of MSCs to GSCXs requires chemoattraction, adhesion to the endothelial wall, and extravasation. Adhesion and extravasation are driven largely by lectin-carbohydrate interactions. Measurements of MSC and GSCX glycogene expression, glycan expression on cell surface proteins, and quantitative proteomics were performed. We compared the molecular landscapes of attracting vs. non-attracting GSCXs. Key findings were the shift from high-mannose type glycosylation on MSCs following exposure to chemoattractants and expression of SIGLEC9 by attractor GSCXs versus expression of SIGLEC8 in non-attractors. Attractor GSCXs displayed down-regulated galactosyltransferase activity, whereas non-attractors were enriched in transcripts related to mannosyltransferases. In attractor GSCXs, kallikrein 4, 13, 14, and 15 were upregulated. These serine proteases are likely to contribute to the modulation of the tumoral extracellular biochemical environment. Results from this project will lead to future engineering strategies to enhance engraftment of MSCs in gliomas, a concept that is likely to have great clinical benefit.

Modeling and analysis of drug-eluting stents with biodegradable PLGA coating: consequences on intravascular drug delivery.

Increasing interests have been raised toward the potential applications of biodegradable poly(lactic-co-glycolic acid) (PLGA) coatings for drug-eluting stents in order to improve the drug delivery and reduce adverse outcomes in stented arteries in patients. This article presents a mathematical model to describe the integrated processes of drug release in a stent with PLGA coating and subsequent drug delivery, distribution, and drug pharmacokinetics in the arterial wall. The integrated model takes into account the PLGA degradation and erosion, anisotropic drug diffusion in the arterial wall, and reversible drug binding. The model simulations first compare the drug delivery from a biodegradable PLGA coating with that from a biodurable coating, including the drug release profiles in the coating, average arterial drug levels, and arterial drug distribution. Using the model for the PLGA stent coating, the simulations further investigate drug internalization, interstitial fluid flow in the arterial wall, and stent embedment for their impact on drug delivery. Simulation results show that these three factors, while imposing little change in the drug release profiles, can greatly change the average drug concentrations in the arterial wall. In particular, each of the factors leads to significant and yet distinguished alterations in the arterial drug distribution that can potentially influence the treatment outcomes. The detailed integrated model provides insights into the design and evaluation of biodegradable PLGA-coated drug-eluting stents for improved intravascular drug delivery.

Intravascular cell delivery device for therapeutic VEGF-induced angiogenesis in chronic vascular occlusion

Site specific targeting remains elusive for gene and stem cell therapies in the cardiovascular field. One promising option involves use of devices that deliver larger and more sustained cell/gene payloads to specific disease sites using the versatility of percutaneous vascular access technology. Smooth muscle cells (SMCs) engineered to deliver high local concentrations of an angiogenic molecule (VEGF) were placed in an intravascular cell delivery device (ICDD) in a porcine model of chronic total occlusion (CTO) involving ameroid placement on the proximal left circumflex (LCx) artery. Implanted SMC were retained within the ICDD and were competent for VEGF production in vitro and in vivo. Following implantation, micro-CT analyses revealed that ICDD-VEGF significantly enhanced vasa vasora microvessel density with a concomitant increase in tissue VEGF protein levels and formation of endothelial cell colonies suggesting increased angiogenic potential. ICDD-VEGF markedly enhanced regional blood flow determined by microsphere and contrast CT analysis translating to a functional improvement in regional wall motion and global left ventricular (LV) systolic and diastolic function. Our data indicate robust, clinically relevant angiogenesis can be achieved in a human scale porcine chronic vascular occlusion model following ICDD-VEGF-based delivery of angiogenic cells. This may have implications for percutaneous delivery of numerous therapeutic factors promoting creation of microvascular bypass networks in chronic vaso-occlusive diseases.

Dispersion characteristics of blood during nanoparticle assisted drug delivery process through a permeable microvessel

Nanoparticle assisted drug delivery holds considerable promise as a means of next generation of medicine that allows for the intravascular delivery of drugs and contrast agents. We analyze the dispersion characteristics of blood during a nanoparticle-assisted drug delivery process through a permeable microvessel. The contribution of molecular and convective diffusion is based on Taylor's theory of shear dispersion. The aggregation of red blood cells in blood flowing through small tubes (less than 40μm) leads to the two-phase flow with a core of rouleaux surrounded by a cell-depleted peripheral layer. The core region models as a non-Newtonian Casson fluid and the peripheral region acts as a Newtonian fluid. We investigate the influence of the nanoparticle volume fraction, the permeability of the blood vessel, pressure distribution, yield stress and the radius of the nanoparticle on the effective dispersion. We show that the effective diffusion of the nanoparticles reduces with an increase in nanoparticle volume fraction. The permeability of the blood vessels increases the effective dispersion at the inlet. The present study contributes to the fundamental understanding on how the particulate nature of blood influences nanoparticle delivery, and is of particular significance in nanomedicine design for targeted drug delivery applications.

Intra-arterial delivery of human bone marrow mesenchymal stem cells is a safe and effective way to treat cerebral ischemia in rats.

Cerebral ischemic stroke is a very common condition that can cause death and disability. Studies have confirmed that stem cells have therapeutic effects if administered after a stroke. There is still a great deal of debate regarding the best route for cell transplantation. Intravascular delivery is the most commonly used one. In this study, the therapeutic effects of bone marrow stem cells (BMSCs) delivered by intra-arterial (IA) and intravenous (IV) injection in a rat transient middle cerebral artery occlusion model (MCAO) are compared. Histological analysis demonstrated that the IA route bypasses the pulmonary system and directs the cells to the ischemic parts of the brain more efficiently. The BMSCs delivered via the IA route promoted angiogenesis and improved functional recovery. The cerebral blood flow (CBF) of the rats was monitored during the IA injection process. No reduction in CBF or microstrokes was detected. Brain perfusion and metabolism, as evaluated by SPECT and PET, were better in rats treated with cells delivered via IA. Results showed that the IA route is a safe and effective way to transplant hBMSCs. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

First study of intra-arterial delivery of myogenic mononuclear cells to skeletal muscles in primates.

The main challenge of cell transplantation as a treatment of myopathies is the large amount of tissue to treat. Intravascular delivery of cells may be an ideal route if proven to be effective and safe. Given the importance of nonhuman primates for preclinical research in transplantation, we tested the intra-arterial injection of β-galactosidase (β-Gal)-labeled myoblasts in macaques. Cells were injected into one of the femoral arteries in seven monkeys. Some muscle sites were damaged concomitantly in three monkeys. Various organs and muscles were sampled 1 h, 1 day, 12 days, 3 weeks, and 5 weeks after transplantation. Samples were analyzed by histology. Most β-Gal(+) cells were observed in the capillaries and arterioles of muscles and other tissues of the leg homolateral to the cell injection. Groups of necrotic myofibers in the proximity of an arteriole plugged by a β-Gal(+) embolus were interpreted as microinfarcts. Scarce β-Gal(+) cells were observed in the lungs 1 h and 1 day posttransplantation. No β-Gal(+) cells were observed in other organs or muscles. β-Gal(+) myofibers were observed 12 days, 3 weeks, and 5 weeks after transplantation in muscles of the leg after the cell injection, in sites that were damaged at the time of cell injection. In conclusion, most intra-arterially injected myoblasts were retained in vessels of the leg homolateral to the cell injection site, and they fused with myofibers in regions in which there was a process of myofiber regeneration. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Concentration rather than dose defines the local brain toxicity of agents that are effectively distributed by convection-enhanced delivery

BackgroundConvection-enhanced delivery (CED) has been developed as a potentially effective drug-delivery strategy into the central nervous system. In contrast to systemic intravenous administration, local delivery achieves high concentration and prolonged retention in the local tissue, with increased chance of local toxicity, especially with toxic agents such as chemotherapeutic agents. Therefore, the factors that affect local toxicity should be extensively studied. New methodWith the assumption that concentration-oriented evaluation of toxicity is important for local CED, we evaluated the appearance of local toxicity among different agents after delivery with CED and studied if it is dose dependent or concentration dependent. ResultsLocal toxicity profile of chemotherapeutic agents delivered via CED indicates BCNU was dose-dependent, whereas that of ACNU was concentration-dependent. On the other hand, local toxicity for doxorubicin, which is not distributed effectively by CED, was dose-dependent. Local toxicity for PLD, which is extensively distributed by CED, was concentration-dependent. Comparison with existing methodTraditional evaluation of drug induced toxicity was dose-oriented. This is true for systemic intravascular delivery. However, with local CED, toxicity of several drugs exacerbated in concentration-dependent manner. From our study, local toxicity of drugs that are likely to distribute effectively tended to be concentration-dependent. ConclusionConcentration rather than dose may be more important for the toxicity of agents that are effectively distributed by CED. Concentration-oriented evaluation of toxicity is more important for CED.

Intracoronary and retrograde coronary venous myocardial delivery of adipose‐derived stem cells in swine infarction lead to transient myocardial trapping with predominant pulmonary redistribution

To examine the comparative fate of adipose-derived stem cells (ASCs) as well as their impact on coronary microcirculation following either retrograde coronary venous (RCV) or arterial delivery. Local delivery of ASCs to the heart has been proposed as a practical approach to limiting the extent of myocardial infarction. Mouse models of mesenchymal stem cell effects on the heart have also demonstrated significant benefits from systemic (intravenous) delivery, prompting a question about the advantage of local delivery. There has been no study addressing the extent of myocardial vs. systemic disposition of ASCs in large animal models following local delivery to the myocardium. In an initial experiment, dose-dependent effects of ASC delivery on coronary circulation in normal swine were evaluated to establish a tolerable ASC dosing range for intracoronary (IC) delivery. In a set of subsequent experiments, an anterior acute myocardial infarction (AMI) was created by balloon occlusion of the proximal left anterior descending (LAD) artery, followed by either IC or RCV infusion of 10(7) (111)Indium-labeled autologous ASCs 6 days following AMI. Indices of microcirculatory resistance (IMR) and coronary flow reserve (CFR) were measured before sacrifices to collect tissues for analysis at 1 or 24 hr after cell delivery. IC delivery of porcine ASCs to normal myocardium was well tolerated up to a cumulative dose of 14 × 10(6) cells (approximately 0.5 × 10(6) cells/kg). There was evidence suggesting microcirculatory trapping of ASC: at unit doses of 50 × 10(6) ASCs, IMR and CFR were found to be persistently altered in the target LAD distribution at 7 days following delivery, whereas at 10 × 10(6) ASCs, only CFR was altered. In the context of recent MI, a significantly higher percentage of ASCs was retained at 1 hr with IC delivery compared with RCV delivery (57.2 ± 12.7% vs. 17.9 ± 1.6%, P = 0.037) but this initial difference was not apparent at 24 hr (22.6 ± 5.5% vs. 18.7 ± 8.6%; P = 0.722). In both approaches, most ASC redistributed to the pulmonary circulation by 24 hr postdelivery. There were no significant differences in CFR or IMR following ASC delivery to infarcted tissue by either route. Selective intravascular delivery of ASC by coronary arterial and venous routes leads to similarly limited myocardial cell retention with predominant redistribution of cells to the lungs. IC arterial delivery of ASC leads to only transiently greater myocardial retention, which is accompanied by obstruction of normal regions of coronary microcirculation at higher doses. The predominant intrapulmonary localization of cells following local delivery via both methods prompts the notion that systemic delivery of ASC might provide similarly beneficial outcomes while avoiding risks of inadvertent microcirculatory compromise.

Removing the Immune Response From Muscular Dystrophy Research

Immune responses against exogenous cells, vectors, and proteins have emerged as a significant barrier limiting development of therapies for inherited disorders. This is especially true when testing human cells and genes in animal models of disease. In this issue of Molecular Therapy, Vallese et al.1 describe an improved immunodeficient mouse model for Duchenne muscular dystrophy (DMD) that should facilitate the development of therapies. The authors show that these mice enable extensive testing of human stem cells and methods of delivery without immune-mediated rejection of donor cells. The utility of this model is further enhanced by use of a specific mutant allele that eliminates background dystrophin expression in muscle tissues, providing a sensitive platform for monitoring cell engraftment and exogenous gene expression. As such, the model could accelerate development of interventions based on both stem cell transplantation and genetic manipulation.

Influence of a paclitaxel coated balloon in combination with a bare metal stent on restenosis and endothelial function: Comparison with a drug eluting stent and a bare metal stent

Different approaches of local intravascular drug delivery may influence endothelial and microvascular function. The aim of this trial was to study the influence of a paclitaxel coated balloon in combination with a bare metal stent (DCB + BMS) versus a bare metal stent (BMS) or a sirolimus-eluting stent (DES) on coronary restenosis and endothelial function. This prospective trial included 77 patients with coronary de novo lesions. The patients were assigned to either one of the treatment groups. After 9 months, patients underwent angiographic follow-up including invasive measurement of coronary endothelial function. After 9 months, late lumen loss in-stent was highest in the BMS group (0.85 ± 0.73 mm), lower in DCB + BMS (0.36 ± 0.46 mm); and lowest in the DES group (0.25 ± 0.34 mm; P = 0.001 [ANOVA]). When compared to the BMS group, in-segment late lumen loss was significantly reduced in the DCB + BMS group (0.27 ± 0.43 mm vs. 0.60 ± 0.55 mm, P = 0.029) and the DES group (0.28 ± 0.40 mm, P = 0.045). Coronary flow reserve was significantly higher with the DCB + BMS treatment (3.16 ± 0.97 vs. 2.42 ± 0.99 [BMS], P = 0.036) whereas the increase in the DES group did not reach the significance level (3.06 ± 1.39, P = 0.144 vs. BMS). Parameters of endothelial function like intracoronary flow velocity and vessel diameter distal to the stented area showed similar patterns of response to adenosine, acetylcholine, and nitro in all groups. DES and the combination of DCB + BMS showed a significant reduction of late lumen loss as compared to a BMS alone. Furthermore, both types of local drug delivery were not associated with a deterioration of microvascular function at 9 months [ClinicalTrials.gov Identifier: NCT00473499].

Binding of an intravascular delivery vehicle for prevention of arterial restenosis is contingent on tertiary structure

CONCLUSIONS: These data suggest that TXA decreases the time to initial clot formation and increases the rate of clot propagation indicating its procoagulant effects beyond fibrinolysis, which are relevant to both potential benefit as well as safety. Thus, it is imperative to confirm a diagnosis of pathologic fibrinolysis prior to treatment with TXA to avoid exposure of a patient to a potentially thrombogenic drug.

Improved wound healing of postischemic cutaneous flaps with the use of bone marrow‐derived stem cells

To determine if the intravascular delivery of mesenchymal stem cells improves wound healing and blood perfusion to postischemic cutaneous flap tissues. Randomized controlled study. A murine model of a cutaneous flap was created based on the inferior epigastric vessels. Mice (n = 14) underwent 3.5 hours of ischemia followed by reperfusion. Bone marrow stromal cells (BMSCs) 1 × 10(6) were injected intravenously. Wound healing was then assessed measuring percent flap necrosis, flap perfusion, and tensile strength of the flap after a period of 14 days. Localization of BMSCs was determined with radiolabeled and fluorescent labeled BMSCs. Postischemic cutaneous flap tissues treated with BMSCs demonstrated significantly less necrosis than control flaps (P <0.01). Beginning on postoperative day 5, BMSC-treated flaps demonstrated greater blood perfusion than untreated flaps (P <0.01). Tensile strength of BMSC-treated cutaneous flaps was significantly higher (P <0.01), with a mean strength of 283.4 ± 28.4 N/m than control flaps with a mean of 122.4 ± 23.5 N/m. Radiolabeled BMSCs localized to postischemic flaps compared to untreated tissues (P = 0.001). Fluorescent microscopy revealed incorporation of BMSCs into endothelial and epithelial tissues of postischemic flaps. This study demonstrates that the intravascular delivery of BMSCs increases wound healing and promotes flap survival following ischemia-reperfusion injury of cutaneous tissue flaps.

Sirolimus-loaded stealth colloidal systems attenuate neointimal hyperplasia after balloon injury: A comparison of phospholipid micelles and liposomes

Restenosis after angioplasty remains a serious complication in clinical cardiology. This study aims to investigate the stealth colloidal systems for local intra-arterial drug delivery. Micelles from polyethylene glycol conjugated with phosphatidylethanolamine and PEGylated liposomes loaded with sirolimus were prepared and characterized with regard to their loading efficiency, particle size distribution, zeta potential, morphology, nuclear magnetic resonance spectroscopy, drug release profile and stability. The antirestenotic effects of the sirolimus-loaded micelles (14 nm) and liposomes (90 nm) were evaluated and compared in the rat carotid injury model following local intravascular delivery. In comparison to control groups, treatment of balloon injured rats with drug loaded micelles and nanoliposomes significantly reduced vascular stenosis by 42% and 19%, respectively (P<0.05). In addition, the luminal area was significantly enlarged by 39% and 60% following treatment with sirolimus-loaded liposomes and micelles, respectively (P<0.05). Immunohistochemistry revealed that sirolimus-loaded nanocarriers suppressed cell proliferation (Ki67-positive cells) as compared to control groups without affecting the density of smooth muscle actin staining. These results suggest that both colloidal nanocarriers could serve as effective intramural drug delivery systems for the treatment of restenosis; however, phospholipid based micelles provided better antirestenotic effects than PEGylated liposomes.

Overexpression of the Mesenchymal Stem Cell Cxcr4 Gene in Irradiated Mice Increases the Homing Capacity of These Cells

The efficiency of the intravascular delivery of mesenchymal stem cells (MSCs) homing to bone marrow has been largely limited. This study aimed to evaluate the homing efficacy in irradiated mice of MSCs that have been engineered to overexpress the murine Cxcr4 gene. Mouse MSCs were infected by a lentivirus vector carrying Cxcr4. MSC migration was detected by an in vitro transwell migration assay. EGFP-positive MSCs were systemically injected into BALB/c mice and detected in bone marrow samples by flow cytometry. The concentration of mouse stromal-derived factor 1 was detected by ELISA. The plasma concentration of the inflammatory cytokines, interleukin (IL)-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70, were determined by cytometric bead array. MSCs that overexpressed Cxcr4 displayed enhanced migration toward a stromal-derived factor 1 gradient. The transplantation of Cxcr4-overexpressing MSCs into irradiated mice leads to increased homing to the bone marrow. Moreover, the frequency of the EGFP-positive cells in a bone marrow infusion 24 h after total body irradiation was 2.2-fold more than at 4 h after irradiation. The concentration of both plasma and bone marrow stromal-derived factor 1 increased after irradiation, and this was positively correlated with the number of Cxcr4-overexpressing MSCs homing to the bone marrow. Moreover, compared with the control groups, the plasma levels of IL-6, IFN-γ, TNF-α, and MCP-1 and IL-12p70 in recipients infused with Cxcr4-overexpressing MSCs was significantly decreased. The level of IL-10 was increased, which correlated with changes in the Th1 and Th2 subset balance. MSCs that overexpressed Cxcr4 and were injected into irradiated mice had an enhanced homing capacity which was related to the bone marrow level of stromal-derived factor 1.

Emerging technologies: polymer-free phospholipid encapsulated sirolimus nanocarriers for the controlled release of drug from a stent-plus-balloon or a stand-alone balloon catheter

Drug-eluting stents have proven to be effective in reducing the risk of late restenosis. In order to achieve a controlled and prolonged release of the antiproliferative agent, current drug-eluting stents utilise various biodegradable as well as non-erodible polymeric blends to coat the stent surface and to serve as drug carriers. The utilisation of polymeric compounds in current drug-eluting stents may eventually limit their performance as well as their clinical applicability due to the potential induction of undesirable local reactions. The development of alternative, polymer-free drug carriers has the potential to overcome some of the limitations of current drug-eluting stent formulations. Moreover, improvements in drug carriers may also result in an expansion of the technological possibilities for other intravascular drug delivery systems, such as metal-free or even implant-free solutions. This article describes the structure and the preclinical validation profile of a novel phospholipid encapsulated sirolimus nanocarrier, used as a coating in two formulations: a coronary stent-plus-balloon system and a stand-alone balloon catheter. The nanoparticles provided a stable, even and homogenous coating to the devices in both formulations. Dose-finding studies allowed the most appropriate identification of the best nanoparticle structure associated with an extremely efficient transfer of drug to all layers of the vessel wall, achieving high tissue concentrations that persisted days after the application, with low systemic drug leaks.

Non-invasive delivery of small interfering ribonucleic acid for reduction of Huntingtin expression in the brain is achieved using focused ultrasound to disrupt the blood-brain barrier

Huntington’s disease is caused by a mutation in the Huntingtin (Htt) gene, which leads to neuronal dysfunction and cell death. Silencing of the Htt gene can halt or reverse the progression of the disease indicating that RNA interference is the most effective strategy for disease treatment. However, small interfering RNA (siRNA) does not cross the blood-brain barrier and therefore delivery to the brain is limited. Here, we demonstrate that focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48 hours following treatment with siRNA, the right (treated) and left (control) striatum was dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients.

“Infectious” Optimism for Treatment of Hepatocellular Carcinoma

“Infectious” Optimism for Treatment of Hepatocellular Carcinoma

Gene Therapy for Canavan's Disease Takes a Step Forward

Gene Therapy for Canavan's Disease Takes a Step Forward

Surface Modification of Coronary Stents for Intravascular Gene Delivery

The present study investigated a novel surface modification on metal coronary stent for antibody immobilization. Methods: 316L stainless steel stents were surface modified with protein coatings. An Anti-DNA antibody was covalently bound to the protein surface using a bi-functional cross-linking agent SPDP. The artwork and binding stability of protein coatings were evaluated by in-vitro eluting. The feasibility and stability of anti-DNA antibody covalently bound to the stent were evaluated by means of 125I labeling. Results: We observed that pre-treating the steel surface using diluted HCL and increasing the ratio of cross-linking agent caused significantly increased binding stability of the protein coatings (p﹤0.001). The amount of chemically coupled antibody on the stents was 8 times higher than that of physically absorbed control stents. The stability of chemically coupled antibody on the stent was significantly better than physically absorbed control. Conclusion: It is concluded that we optimized the technique of protein coating on stainless steel and achieved stable anti-DNA antibody immobilization, therefore enabled efficient and highly localized non-viral gene delivery.

Minimizing the Inhibitory Effect of Neutralizing Antibody for Efficient Gene Expression in the Liver With Adeno-associated Virus 8 Vectors

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.