Therapeutic extracellular vesicle production is substantially increased by inhibition of cellular cholesterol biosynthesis.

Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of the manufacturing process are rich with opportunities to enhance the ability to deliver these medicines to patients. With the goal of improving cell culture EV productivity, we have utilized high throughput siRNA screens to identify the underlying genetic pathways that regulate EV productivity to inform rational host cell line engineering and media development approaches. The screens identified multiple metabolic pathways of potential interest; one of which was validated and shown to be a ready implementable, cost-effective strategy to increase EV titers. We show that both EV volumetric and specific productivity from HEK293 and CHO-S were increased in a dose and cell line-dependent manner up to ninefold when cholesterol synthesis was inhibited by the inclusion of statins in the cell culture media. In addition, we show in response to statin treatment, elevation of EV markers in mesenchymal stem cell (MSC) cell culture media suggesting this approach can also be applicable to MSC EVs. Furthermore, we show that the EVs produced from statin-treated HEK293 cultures are effectively loaded by both endogenous and exogenous loading methods and have equivalent in vitro or in vivo potency relative to EVs from untreated cultures.

Diagnosis and treatment outcomes from prebronchodilator spirometry performed alongside lung cancer screening in a Lung Health Check programme.

INTRODUCTION: Incorporating spirometry into low-dose CT (LDCT) screening for lung cancer may help identify people with undiagnosed chronic obstructive pulmonary disease (COPD), although the downstream impacts are not well described. METHODS: Participants attending a Lung Health Check (LHC) as part of the Yorkshire Lung Screening Trial were offered spirometry alongside LDCT screening. Results were communicated to the general practitioner (GP), and those with unexplained symptomatic airflow obstruction (AO) fulfilling agreed criteria were referred to the Leeds Community Respiratory Team (CRT) for assessment and treatment. Primary care records were reviewed to determine changes to diagnostic coding and pharmacotherapy. RESULTS: Of 2391 LHC participants undergoing prebronchodilator spirometry, 201 (8.4%) fulfilled the CRT referral criteria of which 151 were invited for further assessment. Ninety seven participants were subsequently reviewed by the CRT, 46 declined assessment and 8 had already been seen by their GP at the time of CRT contact. Overall 70 participants had postbronchodilator spirometry checked, of whom 20 (29%) did not have AO. Considering the whole cohort referred to the CRT (but excluding those without AO postbronchodilation), 59 had a new GP COPD code, 56 commenced new pharmacotherapy and 5 were underwent pulmonary rehabilitation (comprising 2.5%, 2.3% and 0.2% of the 2391 participants undergoing LHC spirometry). CONCLUSIONS: Delivering spirometry alongside lung cancer screening may facilitate earlier diagnosis of COPD. However, this study highlights the importance of confirming AO by postbronchodilator spirometry prior to diagnosing and treating patients with COPD and illustrates some downstream challenges in acting on spirometry collected during an LHC.

Transfection reagent artefact likely accounts for some reports of extracellular vesicle function.

Extracellular vesicles (EV) are important mediators of cell communication and physiology. EVs are frequently investigated by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s) or nucleic acid(s) of interest. DNA-transfection reagent complexes (DTC) are approximately the same size as EVs, raising the possibility that some purification procedures may fail to separate these two species and activity arising from carryover DTC may be improperly attributed to EVs. We find that differential ultracentrifugation, a commonly employed EV isolation procedure, does not separate EVs from DTC present in the cell culture supernatant of transiently transfected cells. We demonstrate that the biological activity of an EV-directed Cre recombinase is due to contaminating plasmid DNA and not EV-mediated delivery of Cre protein. Moreover, steps commonly taken to remove plasmid DNA from EV samples, such as media exchanges and treatment with nucleases, are ineffective at avoiding this artefact. Due to the pernicious nature of plasmid DNA in these cellular assays, some reports of EV function are likely artefacts produced by contaminating DTC. EVs and DTC can be separated by density gradient ultracentrifugation, highlighting the importance of validating elimination of DTC when using transient transfection of EV-producing cells to interrogate EV function.

Evolutionary insights into coagulation factor IX Padua and other high-specific-activity variants.

The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy in Nonhuman Primates.

Adeno-associated virus (AAV) vector gene therapy is a promising treatment for a variety of genetic diseases, including hemophilia. Systemic administration of AAV vectors is associated with a cytotoxic immune response triggered against AAV capsid proteins, which if untreated can result in loss of transgene expression. Immunosuppression (IS) with corticosteroids has limited transgene loss in some AAV gene therapy clinical trials, but was insufficient to prevent loss in other studies. We used a nonhuman primate model to evaluate intensive T cell-directed IS combined with AAV-mediated transfer of the human factor IX (FIX) gene. Early administration of rabbit anti-thymocyte globulin (ATG) concomitant with AAV administration resulted in the development of anti-FIX antibodies, whereas delayed ATG by 5 weeks administration did not. The anti-FIX immune response was associated with increases in inflammatory cytokines, as well as a skewed Th17/regulatory T cell (Treg) ratio. We conclude that the timing of T cell-directed IS is critical in determining transgene-product immunogenicity or tolerance. These data have implications for systemically administered AAV gene therapy being evaluated for hemophilia A and B, as well as other genetic diseases.

Hyperactivity of factor IX Padua (R338L) depends on factor VIIIa cofactor activity.

Adeno-associated-viral (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector-dose-dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an eightfold increased specific activity compared to FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor-dependence of FIX-R338L relative to FIX-WT. We observed that the high-specific-activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a novel system utilizing emicizumab, a FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated-transgene-expressed FIX-R338L from HB GT subjects is ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor-dependence of FIX-R338L is similar to FIX-WT, but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.

Longitudinal Deformation of the Right Ventricle in Hypoplastic Left Heart Syndrome: A Comparative Study of 2D-Feature Tracking Magnetic Resonance Imaging and 2D-Speckle Tracking Echocardiography.

In hypoplastic left heart syndrome (HLHS), long-term outcome is closely related to right ventricular function. Echocardiography and magnetic resonance imaging (MRI) are routinely used for functional assessment. MRI 2D-tissue feature tracking (2D-FT) allows quantification of myocardial deformation but has not yet been applied to HLHS patients. We sought to investigate the feasibility of this technique and to compare the results to 2D-speckle tracking echocardiography (2D-STE). In routine MRI 2D anatomical four chamber view, cine images were recorded in 55 HLHS patients (median age 4.9 years [1.6, 17.0]). Regional and global peak systolic longitudinal strain (LS) and strain rate (LSR) were determined using 2D-FT software. Echocardiographic four chamber view was analyzed with 2D-STE. Visualization of all myocardial segments with MRI was excellent, regional, and global LS and LSR could be assessed in all data sets. In 2D-STE, 28% of apical segments could not be analyzed due to poor image quality. Agreement of 2D-FT MRI and 2D-STE was acceptable for global LS, but poor for global LSR. In MRI, regional LS was lower in the septal segments, while LSR was not different between the segments. GLS and GLSR correlated with ejection fraction (GLS: r = - 0.45 and r < 0.001, GLSR: r = - 0.34 and p = 0.01). With new post-processing options, the assessment of regional and global LS and LSR is feasible in routine MRI of HLHS patients. For LS, results were comparable with 2D-STE. The agreement was poor for LSR, which might relate to differences in temporal resolution between the two imaging modalities.

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Empty Capsids and Macrophage Inhibition/Depletion Increase rAAV Transgene Expression in Joints of Both Healthy and Arthritic Mice.

Gene therapy has potential to treat rheumatic diseases; however, the presence of macrophages in the joint might hamper adeno-associated viral vector-mediated gene delivery. Here we demonstrate that in arthritic, but also in healthy, mice administration of agents that influence macrophage activity/number and/or addition of empty decoy capsids substantially improve the efficacy of recombinant adeno-associated viral vector 5 transgene expression in the joint. Pretreatment with triamcinolone or clodronate liposomes improved luciferase expression over a period of 4 weeks. Similar results were seen when empty decoy capsids were added to full genome containing capsids in a 5:1 ratio. In a study to assess the duration of expression as well as to investigate the combination of these two approaches, we observed a synergistic enhancement of gene expression, sustained for at least 12 weeks. The enhancement of gene expression was independent of the route of administration of triamcinolone (intra-articular or intramuscular). In healthy mice it was demonstrated that the combination improved expression of the transgene significantly, in a serotype independent manner. These data have implications for future applications of gene therapy to the joint and for other tissues with an abundance of macrophages.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB.

Characterization of naturally-occurring humoral immunity to AAV in sheep.

AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.

Recent clinical trials have shown that evasion of CD8(+) T-cell responses against viral capsid is critical for successful liver-directed gene therapy with adeno-associated viral (AAV) vectors for hemophilia. Preclinical models to test whether use of alternate serotypes or capsid variants could avoid this deleterious response have been lacking. Here, the ability of CD8(+) T cells ("cap-CD8," specific for a capsid epitope presented by human B*0702 or murine H2-L(d) molecules) to target AAV-infected hepatocytes was investigated. In a murine model based on adoptive transfer of ex vivo expanded cap-CD8, AAV2-transduced livers showed CD8(+) T-cell infiltrates, transaminitis, significant reduction in factor IX transgene expression, and loss of transduced hepatocytes. AAV8 gene transfer resulted in prolonged susceptibility to cap-CD8, consistent with recent clinical findings. In contrast, using an AAV2(Y-F) mutant capsid, which is known to be less degraded by proteasomes, preserved transgene expression and largely avoided hepatotoxicity. In vitro assays confirmed reduced major histocompatibility complex class I presentation of this capsid and killing of human or murine hepatocytes compared with AAV2. In conclusion, AAV capsids can be engineered to substantially reduce the risk of destruction by cytotoxic T lymphocytes, whereas use of alternative serotypes per se does not circumvent this obstacle.

The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy.

Studies on gene therapy for hemophilia B (HB) using adeno-associated viral (AAV) vectors showed that the safety of a given strategy is directly related to the vector dose. To overcome this limitation, we sought to test the efficacy and the risk of immunogenicity of a novel factor IX (FIX) R338L associated with ∼ 8-fold increased specific activity. Muscle-directed expression of canine FIX-R338L by AAV vectors was carried out in HB dogs. Therapeutic levels of circulating canine FIX activity (3.5%-8%) showed 8- to 9-fold increased specific activity, similar to humans with FIX-R338L. Phenotypic improvement was documented by the lack of bleeding episodes for a cumulative 5-year observation. No antibody formation and T-cell responses to FIX-R338L were observed, even on challenges with FIX wild-type protein. Moreover, no adverse vascular thrombotic complications were noted. Thus, FIX-R338L provides an attractive strategy to safely enhance the efficacy of gene therapy for HB.

In vivo genome editing restores haemostasis in a mouse model of haemophilia.

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.

CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.

We have recently reported that CD8(+) T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8(+) T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8(+) T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8(+) T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

Safety of liver gene transfer following peripheral intravascular delivery of adeno-associated virus (AAV)-5 and AAV-6 in a large animal model.

Intravascular delivery of adeno-associated virus (AAV) vector is commonly used for liver-directed gene therapy. In humans, the high prevalence of neutralizing antibodies to AAV-2 capsid and the wide cross-reactivity with other serotypes hamper vector transduction efficacy. Moreover, the safety of gene-based approaches depends on vector biodistribution, vector dose, and route of administration. Here we sought to characterize the safety of AAV-5 and AAV-6 for liver-mediated human factor IX (hFIX) expression in rabbits at doses of 1 × 10(12) or 1 × 10(13) viral genomes/kg. Circulating therapeutic levels of FIX were observed in both cohorts of AAV-6-hFIX, whereas for AAV-5-hFIX only the high dose was effective. Long-lasting inhibitory antibodies to hFIX were detected in three of the 10 AAV-6-injected animals but were absent in the AAV-5 group. Overall, vector shedding in the semen was transient and vector dose-dependent. However, the kinetics of clearance were remarkably faster for AAV-5 (3-5 weeks) compared with AAV-6 (10-13 weeks). AAV-6 vector sequences outside the liver were minimal at 20-30 weeks post-injection. In contrast, AAV-5 exhibited relatively high amounts of vector DNA in tissues other than the liver. Together these data are useful to further define the safety and potential for clinical translation of these AAV vectors.

Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy.

Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

Proteasome inhibitors decrease AAV2 capsid derived peptide epitope presentation on MHC class I following transduction.

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.