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. 2022 Feb 14;33(3-4):119–130. doi: 10.1089/hum.2021.083

Dexamethasone Transiently Enhances Transgene Expression in the Liver When Administered at Late-Phase Post Long-Term Adeno-Associated Virus Transduction

Zheng Chai 1,†, Xintao Zhang 1,†, Amanda Lee Dobbins 1, Richard Jude Samulski 1,2, Elizabeth P Merricks 3,4, Timothy C Nichols 3,4, Chengwen Li 1,5,6,*
PMCID: PMC8885437  PMID: 34617445

Abstract

Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.

Keywords: adeno-associated virus, liver transduction, glucocorticoid

INTRODUCTION

Viral vector-based gene therapy has been developed and applied for many diseases. Adeno-associated virus (AAV), a non-pathogenic virus, has been used as one of the most efficient viral vectors for rare disease gene therapy because of its broad tissue tropism, low immunogenicity, and overall safety.1,2 At present, 13 AAV serotypes have been identified and some of them have been used in clinical trials, including AAV1, 2, 5, 6, 8, 9, and rh10.1,3,4 Most of the commonly used AAV serotype vectors have a liver tropism.5 Notably, there are two AAV-based gene therapy products approved by the Food and Drug Administration (FDA) in the United States.6,7 One product is for the treatment of an inherited vision loss using AAV2 as a vector to deliver the RPE65 gene, whereas the other is for spinal muscular atrophy treatment using AAV9 to deliver the SMN gene.

The liver plays a critical role in regulating metabolism and other physiological functions.8 Since hepatocytes synthesize and metabolize several proteins involved in metabolism, hemostasis, and detoxification, many inherited metabolic disorders originate in the liver. More than 400 rare monogenic disorders are associated with the liver.9,10 Because of the unique anatomic properties, most AAV vectors delivered systemically accumulate rapidly in the liver, which makes AAV an excellent vector for liver targeted gene delivery. AAV-based gene therapy has been applied for various liver disorders, such as glycogen storage disease, familial hypercholesterolemia, ornithine transcarbamylase deficiency, alpha-1 antitrypsin deficiency, and hemophilia.11 Some limitations and challenges have arisen, however, from the application of AAV vectors for liver targeting in recent clinical trials. Namely, AAV vector administration has been noted to induce immune response against the AAV capsid or transgene and is believed to cause liver toxicity. The toxicity has ultimately resulted in reduce transgene expression and activity.4,12 In light of these clinical observations, preclinical studies have reported that the liver also plays a vital role in inducing tolerance after AAV liver-directed gene therapy.9,13 The observation is confounded by the fact that underlying diseases in the liver appear to potentiate the risk of liver-related toxicities in AAV-based gene therapy. For example, three patients with X-linked myotubular myopathy who originally had progressive liver dysfunction eventually succumbed to death after receiving a high dose of AAV vectors. Unfortunately, clinical data have documented that for liver targeted gene delivery, much higher doses of AAV vectors are needed when compared with animal models.

Regardless, it is well known that glucocorticoids, such as prednisone and dexamethasone, have a potent anti-inflammatory and immunosuppressive function. Glucocorticoids have been used in several liver diseases, most commonly in autoimmune hepatitis for improving outcomes and survival.14 In addition, glucocorticoids are also used for liver transplantation to prevent rejection. Since liver damage occurs in some patients who receive systemic administration of AAV vectors, the use of glucocorticoids has been proposed for preventing liver toxicities.15,16

As seen in the past decades, correcting hemophilia deficiency has been one of the most successful examples of AAV liver-directed gene therapy. AAV-based gene therapy has been extensively studied in preclinical models with both hemophilia A and B deficiency. These animal studies have provided the preclinical data necessary to support and design clinical trials now showing promising results.16–19 Although long-term therapeutic efficacy has been achieved with AAV vectors in classic mouse and dog models of hemophilia A and B, some patients with hemophilia have demonstrated a late-term decline in clotting factor expression and activity.16,20 Unexpectedly, patients who were treated with high-dose AAV vectors have shown a decreased expression level of factor IX (FIX) at week 6 to 10, with a concomitant increase in ALT levels. Ironically, on treatment with prednisone, FIX expression was restored to the previous level. The mechanism underlining this clinical phenomenon is currently unclear. However, some hypothesize that prednisone blocks the immune response activation triggered by the administration of high-dose AAV vectors. We have previously supported this hypothesis and demonstrated that the innate immune response is activated in human hepatocytes during long-term AAV transduction.21

More importantly, in our previous study, we found that mice pretreated with dexamethasone showed a decrease in the global transduction and an increase in the liver transduction of AAV9 vector.22 This outcome would predict specific consequences (i.e., lower levels of systemic vector transduction vs. increase in liver specific transduction) to clinical designs that have implemented steroid pretreatment (e.g., 24-h pre and onward) for muscle or brain, versus liver gene therapy. Based on our previous study, we investigated the effects of dexamethasone on transgene expression in transduced liver after long-term AAV administration, examining (1) time points for administration of dexamethasone post-AAV injection, (2) re-administration of dexamethasone, and (3) duration of dexamethasone application in different animal models, such as wild-type mice, hemophilia mice, and dog models. Our results showed that transgene expression was transiently increased after the application of dexamethasone in mice after long-term AAV transduction, regardless of the duration, timing, or repeated administrations. However, this phenomenon was not reproduced in dogs even though high doses of dexamethasone were used, drawing attention to the selection of animal models for mimicking clinical outcomes. Overall, the study could provide valuable insight for rationally using steroids to regulate transgene expression in future clinic trials.

MATERIALS AND METHODS

Recombinant AAV virus production

Recombinant AAV9 was produced by a triple-plasmid transfection system, as previously described.23 A 15-cm dish of HEK293 cells was transfected with 9 μg of the AAV transgene plasmid, self-complementary (sc)-transthyretin (TTR)-mvm-hFIX, 12 μg of the AAV helper plasmid containing AAV Rep and Cap genes, and 15 μg of the Ad helper plasmid, pXX6–80. Forty-eight hours post-transfection, HEK293 cells were collected and lysed. The supernatant was subjected to a CsCl gradient ultra-centrifugation. Virus titer was determined by quantitative PCR (qPCR). The recombinant AAV9/cFIX vectors were produced by UNC Vector Core for administering hemophilia B dogs.

Animal study

Mice

Animal experiments performed in this study were conducted with wild-type C57BL/6, BALB/c mice and a hemophilia B mouse model in a C57BL/6 background. The mice were maintained in accordance with NIH guidelines, as approved by the UNC Institutional Animal Care and Use Committee (IACUC). Dexamethasone (0.2 mg/mouse) or phosphate-buffered saline (PBS) was injected into mice via intraperitoneal (i.p.) injection at 2 h before the mice received the AAV9/hFIX vectors at a dose of 1 × 1010 vector genomes (vg)/mouse via retro-orbital injection. The same doses of dexamethasone were further injected for different periods of time (1 day, 3 days, or 7 consecutive days) at a later phase post AAV administration. At the same time, PBS was injected into the control mice. At various time points, blood was collected from the retro-orbital plexus.

Dogs

Two hemophilia B dogs, P43 and P44, were maintained at the Francis Owen Blood Research Laboratory at UNC. The dogs were administered AAV9/cFIX at the dose of 1 × 1012 vg/kg body weight. The details are shown in Supplementary Table S2. One year after AAV injection, the dogs were administered dexamethasone at a dose of 1 mg/kg body weight daily for 3 consecutive days. Sera were collected at different time points as indicated. Sixteen weeks after dexamethasone administration, the dogs were re-administered with dexamethasone at a dose of 10 mg/kg body weight daily for 3 consecutive days. Sera were collected, and cFIX protein was measured with canine FIX-specific enzyme-linked immunosorbent assay (ELISA) assay.

Human FIX-specific ELISA assay

Blood was collected from treated mice, as previously described.22 Human FIX-specific ELISA was performed according to the manufacturer's instruction. Briefly, the 96-well plate was coated with anti-human FIX antibody (AHIX-5041, Haematologic Technologies) at a concentration of 2 μg/mL and was kept overnight at 4°C. Then, the coated plate was washed three times and the diluted mice sera and standards were added. After 2 h, the plate was washed three more times, and the horseradish peroxidase conjugated sheep anti-human FIX antibody (SAFIX-APHRP, Affinity Biologics) was added for 1 h. Finally, the plate was washed, and 2, 2'-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] (ABST) was added. After 10 min, the plate was read at a wavelength of 405 nm. The hFIX protein concentrations were calculated according to the manufacturer's protocol.

Canine FIX-specific ELISA assay

Blood was collected from treated hemophilia B dogs, as previously described.24 Canine FIX-specific ELISA was performed as described next. Briefly, the 96-well plate was coated with the capture antibody (CL20044K-C, CEDARLANE) overnight at 4°C. The plate was washed three times, and then the canine sera were diluted and added into the plate for 1 h. After the plate was washed three times, the diluted detecting antibody (CL20044K-D) was added and incubated for 1 h. Finally, the plate was washed three times, and ABST was added. After 10 min, the plate was read at a wavelength of 405 nm. The cFIX protein concentrations were calculated according to the manufacturer's protocol.

RNA isolation and qPCR

The RNA from the mouse liver was isolated by using TRIzol Reagent (15596026, Invitrogen). The first-strand cDNA was synthesized by using the RevertAid First-Strand cDNA Synthesis Kit (K1621; Thermo Fisher Scientific). The qPCR assay was performed and analyzed by using a LightCycler 480 instrument (Roche).

Statistical analysis

The data were presented as mean ± standard deviation. The Student's t-test and two-way analysis of variance (ANOVA) were used for analysis. p Values of <0.05 were considered a statistically significant difference.

RESULTS

Pretreated dexamethasone increased and sustained long-term transduction in the livers of wild-type C57BL/6 mice

In the previous study, we found that the mouse liver transduction from AAV9 vectors encoding firefly luciferase (AAV9/luc) was immediately increased when dexamethasone was injected before AAV9/luc vector administration.22 To explore whether pretreated dexamethasone could sustain the enhancement of liver transduction for other transgenes, we packaged the human FIX gene driven by a liver-specific promoter TTR into the AAV9 vectors (AAV9/hFIX). Dexamethasone or PBS was injected into the wild-type C57BL/6 mice 2 h before AAV9 vector administration. The mice were treated daily with the same dose of dexamethasone for the next 6 days (Fig. 1A). The human FIX-specific ELISA assay was used to evaluate FIX expression in the mice. The results showed that at 1 and 2 weeks post-AAV injection, the FIX expression was significantly higher in the mice with dexamethasone pretreatment than that of the control mice with PBS pretreatment. Notably, the increased FIX expression was also observed at 6 weeks (Fig. 1B). The results suggest that the pretreated dexamethasone increased and maintained the high transgene expression for a long period.

Figure 1.

Figure 1.

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The long-term expression of hFIX in wild-type C57BL/6 mice pretreated with dexamethasone. (A) The schematic of the dexamethasone and AAV9/hFIX administrations and serum collection from blood. Dexamethasone (0.2 mg/mouse) or PBS was injected 2 h before 1 × 1010 vg of AAV9/hFIX vectors per mouse were administered through the retro-orbital vein. The same dose of dexamethasone was administered daily for the next 6 days. The sera were collected at 1, 2, and 6 weeks (W1, W2, and W6), separately. (B) The hFIX expression was measured by human FIX specific ELISA assay. The data represent the average and SD from five mice. The asterisk indicates the significant difference (***p < 0.001 and **p < 0.01). AAV9, adeno-associated virus 9; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; SD, standard deviation; vg, vector genomes. Color images are available online.

FIX expression was increased in wild-type C57BL/6 mice with dexamethasone treatment at late-time post AAV administration

To explore whether the dexamethasone administration at the late time point post-AAV liver targeting could affect the FIX expression, AAV9/hFIX vectors at a dose of 1 × 1010 vg/mouse were administered into wild-type C57BL/6 mice. At 6 weeks post-AAV vector administration, the mice were injected with dexamethasone or PBS for 7 consecutive days. Serum was collected before dexamethasone treatment (6 weeks) and 1 day after the last dose of dexamethasone treatment (7 weeks 1 day). The results showed that the FIX expression increased after dexamethasone application (Fig. 2). The data suggest that dexamethasone administration at a late time point could increase transgene expression from AAV9 vector transduced liver in mice.

Figure 2.

Figure 2.

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The dexamethasone treatment at the late time point increased the hFIX expression in the wild-type C57BL/6 mice. (A) The schematic of the dexamethasone and AAV9/hFIX administrations and serum collection. Two groups (five mice/group) of mice were injected with AAV9/hFIX at a dose of 1 × 1010 vg/mouse. Six weeks later, one group of the mice was administered with dexamethasone (0.2 mg/mouse), whereas the other group of mice was administered with PBS. (B) The hFIX expression in the mice before and after dexamethasone treatment. The sera were collected at 6 weeks and at 7 weeks and 1 day, separately. The hFIX expression was measured by human FIX-specific ELISA assay. The data represent the average and SD from five mice. p > 0.05. The asterisk indicates the significant difference (**p < 0.01). ns, no difference. Color images are available online.

Re-administration of dexamethasone enhanced hFIX expression in wild-type C57BL/6 mice

Since dexamethasone treatment at both the early and late time points enhanced transgene expression, we further investigated the effect of dexamethasone re-administration on transgene expression from AAV vector transduction. As shown in Fig. 3A, a total of 10 mice were pretreated with dexamethasone 2 h before AAV9/hFIX vector administration. The same dose of dexamethasone was administered daily for the next 6 days. At 6 weeks, five mice were re-administered with dexamethasone for 7 consecutive days, whereas the other five mice were treated with PBS. The mice with re-administered dexamethasone had much higher transgene expression than the mice with PBS treatment (Fig. 3B). Most importantly, the transgene expression was also much higher than it was before re-administration (Fig. 3B).

Figure 3.

Figure 3.

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Re-administration of dexamethasone increased the hFIX expression in the wild-type C57BL/6 mice. (A) The schematic of the dexamethasone and AAV9/hFIX administration and serum collection from blood. Dexamethasone (0.2 mg/mouse) or PBS was injected 2 h before 1 × 1010 vg of AAV9/hFIX vectors per mouse were administered through the retro-orbital vein. The same dose of dexamethasone or PBS was administered daily for the next 6 days. Six weeks later, dexamethasone (0.2 mg/mouse) or PBS was re-administered into mice that were pretreated with dexamethasone for 7 days. (B) The hFIX expression in the mice with re-administered dexamethasone. The sera were collected at 7 weeks and 1 day. The hFIX expression was measured by human FIX-specific ELISA assay. The data represent the average and SD from five mice. The asterisk indicates the significant difference (***p < 0.001). FIX, factor IX. Color images are available online.

Re-administrated duration does not affect the effect of dexamethasone on transgene expression increase

The study cited earlier demonstrated that the FIX expression significantly increased after dexamethasone treatment for 7 consecutive days. Next, we tested whether the duration of administration could affect the FIX expression. The result showed that dexamethasone was administered daily at 12 or 18 weeks for 3 consecutive days or 1 day, respectively, in the mice pretreated with dexamethasone (Fig. 4A). An increased expression of hFIX was observed in dexamethasone-treated mice in both regimes (Fig. 4B). These results demonstrated that dexamethasone treatment could increase the expression of hFIX in the wild-type mice independent of the duration of treatment. In addition, the enhanced FIX expression levels returned to the prior level 7 days after the last dose of dexamethasone (Fig. 4B), indicating the transient effect of dexamethasone. This result also implicates that the enhanced transgene expression from dexamethasone treatment can be achieved repeatedly.

Figure 4.

Figure 4.

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The expression of hFIX in the wild-type C57BL/6 mice with different durations of dexamethasone re-administration. (A) The schematic of the dexamethasone and AAV9/hFIX administration and serum collection from blood. Dexamethasone (0.2 mg/mouse) was injected 2 h before 1 × 1010 vg of AAV9/hFIX vectors per mouse was administered through the retro-orbital vein. The same dose of dexamethasone was administered daily for the next 6 days. At 6, 11, and 18 weeks post-AAV injection, dexamethasone (0.2 mg/mouse) or PBS was re-administered consecutively for 7 days, 3 days, and 1 day, respectively, in the mice pretreated with dexamethasone. (B) The hFIX expression in the mice with different times of dexamethasone re-administration. The time points of the sera collection were indicated in the figure. The data represent the average and SD from five mice. p > 0.05. The asterisk indicates the significant difference (***p < 0.001 and **p < 0.01). Color images are available online.

Administration of dexamethasone increased transgene expression in wild-type C57BL/6 mice with both a low and high dose of AAV9 vectors

To study the effect of dexamethasone on transgene expression from different doses of AAV vectors, we first injected mice with AAV9/hFIX vectors at either 1 × 109 vg/mouse (low dose, Fig. 5A) or 1 × 1011 vg/mouse (high dose, Fig. 5B). At 6 weeks post-AAV injection, half the number of mice with each dose were treated daily with dexamethasone or PBS for 7 consecutive days. The FIX expression was detected one day after the last dose of dexamethasone administration. Consistently, much higher levels of FIX were detected in the mice treated with dexamethasone than in the mice treated with PBS. This result indicates that the enhancement from dexamethasone treatment can be achieved irrespective of the AAV vector dose administered.

Figure 5.

Figure 5.

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The effect of dexamethasone on transgene expression in mice treated with different doses of AAV9/hFIX vectors. The mice were injected with AAV9/FIX vectors at a dose of 1 × 109 vg/mouse (low dose) or 1 × 1011 vg/mouse (high dose), separately. Six weeks later, the mice were treated with dexamethasone (0.2 mg/mouse) or PBS daily for 7 consecutive days. The serum was collected at 7 weeks and 1 day after the last dexamethasone treatment. The hFIX expression was measured by human FIX-specific ELISA assay. (A) The hFIX expression in the mice administered with a low dose of AAV vector. (B) The hFIX expression in the mice administered with a high dose of AAV vector. The data represent the average and SD from five mice. The asterisk indicates the significant difference (***p < 0.001 and *p < 0.05).

FIX expression was increased in BALB/c mice with dexamethasone treatment at late-time post-AAV administration

To explore whether the enhanced transgene expression after dexamethasone treatment is not mouse strain specific, we applied the same approach to evaluate the FIX expression in BALB/c mice with a late-time dexamethasone treatment (Fig. 6A). The BALB/c mice were administered AAV9/hFIX vectors at a dose of 1 × 1010 vg/mouse. After 6 weeks, one dose of dexamethasone was injected. One day later, the sera were collected for FIX ELISA assay. The results showed that hFIX protein expression was significantly increased (Fig. 6B). One week later, we applied dexamethasone for 7 consecutive days and blood was harvested at days 2, 4, and 8 after the first dose of dexamethasone, for FIX expression detection. Consistent with the data in C57BL/6 mice (Fig. 4B), the enhanced hFIX protein expression was observed at any time point after administration of dexamethasone (Fig. 6C). These results support that the augmented transgene expression with dexamethasone is not mouse strain specific.

Figure 6.

Figure 6.

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The effect of dexamethasone on transgene expression in BALB/c mice. (A) The schematic of the dexamethasone and AAV9/hFIX administration and serum collection from blood. The mice were injected with AAV9/FIX vectors at a dose of 1 × 1010 vg/mouse. Six weeks later, the serum was collected and then the mice were treated with one dose of dexamethasone (0.2 mg/mouse). At the 7 weeks, the mice were treated with dexamethasone (0.2 mg/mouse) for 7 consecutive days. The sera were collected at 7 weeks as well as 1, 3, and 7 days after the daily dexamethasone administration. The hFIX expression was measured by human FIX-specific ELISA assay. (B) The hFIX expression at 6 weeks and 1 day. (C) The hFIX expression after the mice were treated with different durations of dexamethasone re-administration. The data represent the average and SD from five mice. The asterisk indicates the significant difference (**p < 0.01 and *p < 0.05). Color images are available online.

Administration of dexamethasone enhances hFIX expression in hemophilia B mouse model

To explore the application of dexamethasone in a potential clinical setting, we studied the effect of dexamethasone on transgene expression from the AAV-transduced liver in a hemophilia B mouse model. We first treated the mice with dexamethasone or PBS through an intraperitoneal injection 2 h before AAV9/hFIX was administered. The same dose of dexamethasone was administered daily for the next 6 days (Fig. 7A). In hemophilia B mice pretreated with dexamethasone, the expression levels of hFIX immediately increased at one day post-AAV injection and were significantly higher than those in mice without dexamethasone treatment (Fig. 7B). At 16 weeks, we re-administered either dexamethasone or PBS in the mice pretreated with dexamethasone for 7 days. The expression levels of hFIX immediately increased as predicted (Fig. 7C). These observations were consistent with the results in the wild-type C57BL/6 mice (Figs. 1 and 2), indicating that the dexamethasone treatment enhanced the transgene expression in both wild-type and hemophilia B mice models.

Figure 7.

Figure 7.

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The effect of dexamethasone on transgene expression in the hemophilia B mice. (A) The schematic of the dexamethasone and AAV9/hFIX administration and serum collection from blood. The hemophilia B mice were treated with dexamethasone (0.2 mg/mouse) or PBS 2 h before 1 × 1010 vg of AAV9/hFIX vectors per mouse was administered through the retro-orbital vein. The same dose of dexamethasone was administered daily for the next 6 days. At 16 weeks post-AAV injection, the mice were re-administered with PBS or dexamethasone for 7 consecutive days. The serum was collected at the different time points. (B) The hFIX expression at 1 week after AAV injection with dexamethasone or PBS administration was measured by human FIX-specific ELISA assay. (C) The hFIX expression after the re-administration of dexamethasone in the pre-dexamethasone treated group was measured by human FIX-specific ELISA assay. The data represent the average and SD from five mice. The asterisk indicates the significant difference (**p < 0.01 and *p < 0.05). Color images are available online.

Dexamethasone did not affect the expression of cFIX in hemophilia B dogs

To investigate whether dexamethasone treatment also enhanced transgene expression from the AAV-transduced liver in larger animals, we treated two hemophilia B dogs P43 and P44 with a systemic injection of AAV9 vectors encoding canine FIX (cFIX) driven by the TTR promoter at a dose of 1 × 1012 vg/kg body weight. About 1 year later, the dogs were injected daily for 3 consecutive days with dexamethasone at a dose of 1 mg/kg body weight. The sera were collected from these two dogs at several time points: before dexamethasone treatment, as well as 1 day, 3 days, 1 week, and 2 weeks after the last administration of dexamethasone. There was no difference in cFIX expression levels among the different time points (Data not shown). To investigate whether the dose of dexamethasone affected transgene expression in hemophilia B dogs 16 weeks after the first treatment of dexamethasone, the dogs were re-administered with 10 mg/kg dexamethasone daily for 3 days. Again, no change was found in cFIX levels in the blood after the dexamethasone treatment (Fig. 8). These observations indicated that the dexamethasone treatment has no impact on cFIX expression from the AAV vector transduced liver in hemophilia B dogs.

Figure 8.

Figure 8.

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The canine FIX expression in the hemophilia B dogs. (A) The schematic of the dexamethasone and AAV9/cFIX administration, and serum collection from blood. Two hemophilia B dogs, P43 and P44 were administered with AAV9/cFIX at a dose of 1 × 1012 vg/kg body weight, separately. One year later, dogs were first administered with dexamethasone at a low dose (1 mg/kg) for consecutive 3 days. At the same time, the sera were collected. After 16 weeks post-dexamethasone, the dogs were re-administered dexamethasone at a high dose (10 mg/kg) for another consecutive 3 days. The sera were collected at different time points. (B) The cFIX proteins were measured with the canine FIX-specific ELISA kit. Color images are available online.

The effect of a high dose of dexamethasone on blood parameters in dogs

We have routinely monitored the liver and kidney functions in hemophilia dogs treated with AAV vectors. Treatment with a low dose of dexamethasone at 1 mg/kg did not induce liver and kidney dysfunction (Data not shown). When a high dose of dexamethasone (10 mg/kg) was used, some parameters related to liver or kidney function including alkaline phosphatase (ALK PHOS), serum glutamyl pyruvic transaminase (SGPT), serum glutamyl oxaloacetic transaminase (SGOT), total bilirubin (Tot Bili), gamma glutamyl transpeptidase (GGTP), and blood urea nitrogen/creatinine (BUN/Creat) were transiently increased (Supplementary Table S1). Most of these parameters returned to normal levels 4 weeks after administration of dexamethasone, and some returned within 2 weeks (Supplementary Table S1).

Dexamethasone treatment decreased the cytokine expression in the livers of the mice

Dexamethasone has immune suppressive function. To explore the effect of dexamethasone on the immune response in the AAV-transduced liver, we detected the interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) mRNA levels in the wild-type C57BL/6 mice treated with AAV and dexamethasone. Mice were pretreated with dexamethasone 2 h before administration of AAV9/hFIX vectors. After 2 h post-AAV vector administration, the livers were taken. The mRNA levels of both IFN-β and TNF-α in the livers from the mice pretreated with dexamethasone were much lower than those in the mice treated with PBS (Fig. 9A). In the following experiment, we first injected the mice with AAV9/hFIX vectors. Six weeks later, the mice were treated with PBS or dexamethasone (0.2 mg/mouse). The livers were harvested 2 h after the dexamethasone or PBS treatment. The mRNA levels of IFN-β in the mice treated with dexamethasone were about three-fold lower than those in the mice treated with PBS (Fig. 9B), which was similar to the results in the liver of mice pretreated with dexamethasone before AAV vector administration (Fig. 9A). It is interesting to note that the mRNA levels of TNF-α decreased drastically in the livers of the mice treated with dexamethasone (Fig. 9B). Based on these results, the treatment of dexamethasone is shown to significantly inhibit the expression of IFN-β and TNF-α in the AAV-transduced livers of mice.

Figure 9.

Figure 9.

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The cytokine expression in AAV-transduced livers of the mice with dexamethasone treatment. (A) The mRNA levels of mouse IFN-β and TNF-α in the liver of the mice that were pretreated with PBS or dexamethasone (0.2 mg/mouse) 2 h before AAV9/hFIX injection. At 2 h post-AAV injection, the livers were taken. (B) The mRNA levels of mouse IFN-β and TNF-α in the liver of the mice with dexamethasone post-treatment at the late time point. The mice were injected with AAV9/hFIX vectors. Six weeks later, the mice were treated with PBS or dexamethasone (0.2 mg/mouse). The livers were taken at 2 h post-dexamethasone. The data represent the average and SD from five mice. The asterisk indicates the significant difference (***p < 0.001, **p < 0.01, and *p < 0.05). IFN-β, interferon beta; TNF-α, tumor necrosis factor alpha.

Dexamethasone treatment at late-time post AAV administration did not have effect on transcription and genome copy number of the transgene

To explore whether the increased transgene expression with dexamethasone treatment was related to transcripts of the transgene or genome copy number in the AAV-transduced liver, we administered dexamethasone once at 6 weeks post AAV/hFIX injection in C57BL mice and harvested the blood and the livers one day later (Fig. 10A). Similar to the previous results, high FIX level was detected in the blood (Fig. 10B). For evaluating the transgene expression at the transcription level and AAV genome copy numbers, we extracted RNA and DNA from the liver. Surprisingly, the treatment with dexamethasone did not induce any change in transgene expression at either the transcriptional level or AAV genome copy number in the AAV-transduced liver (Fig. 10C and D).

Figure 10.

Figure 10.

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No effect of the dexamethasone treatment on transcription and gene copy number of transgene in the C57BL/6 mice. (A) The schematic of the dexamethasone and AAV9/hFIX administration and serum collection from blood. The mice were injected with AAV9/FIX vectors at a dose of 1 × 1010 vg/mouse. Six weeks later, the serum was collected, and the mice were treated with one dose of dexamethasone (0.2 mg/mouse) or PBS. The genomic DNA and RNA were extracted from the livers of the mice. (B) The hFIX expression after the dexamethasone administration was measured by ELISA assay. (C) The mRNA level of hFIX transcription in the mouse livers was measured by qPCR assay. (D) The gene copy number of hFIX in the mouse liver was evaluated by qPCR assay. The data represent the average and SD from five mice. The asterisk indicates the significant difference (**p < 0.01). qPCR, quantitative PCR. Color images are available online.

DISCUSSION

Glucocorticoids are well known for their anti-inflammatory and immunosuppressive functions. Therefore, glucocorticoids, such as prednisolone and dexamethasone, have been used in treating inflammation and preventing rejection reactions.25,26 Dexamethasone has been applied for patients with muscular disorders and central nervous system diseases after systemic administration of AAV vectors for the prevention or treatment of liver damage. However, the effect of dexamethasone on AAV transduction in animals has not been elucidated. In the previous study, we observed that the transgene expression in the AAV vector transduced liver surged with an early dexamethasone treatment.22 In this study, we studied the effect of dexamethasone on AAV transduction when applied at later time points in both mice and dogs. In mice, treatment with dexamethasone was able to transiently increase transgene expression at later time points after AAV administration. The increased transgene expression was not dependent on the duration or time point of dexamethasone administration after AAV injection. Inconsistent with the results in mice, an increased transgene expression was not observed in hemophilia dogs treated with dexamethasone after AAV gene therapy. The mechanism studies showed that the dexamethasone treatment did not change the transcription and genome copy numbers of the transgene. Administration of dexamethasone, however, inhibited the innate immune response and proinflammatory cytokine production in the AAV-transduced livers of mice. Therefore, the likely explanation is that the transiently enhanced transgene expression might be relative to the immunosuppressive function of dexamethasone.

In hemophilia B clinical trials, the administration of glucocorticoids can rescue decreasing transgene expression from liver damage at later time points after AAV vector liver targeted gene therapy. This phenomenon has not been explored in animal models. Consistent with our previous study with luciferase as a transgene,22 pretreatment with dexamethasone resulted in much higher transgene FIX expression in C57BL/6 mice after systemic administration of AAV9/hFIX vectors. When dexamethasone was used at later time points after AAV application, higher transgene expression was also observed. This increased transgene expression was seen when dexamethasone was administered various times (3 or 7 times). However, the increased transgene expression was transient and returned to its previous level within 1 week of the last dose of dexamethasone. The transient increase of transgene expression after dexamethasone treatment is of clinical significance, especially for hemophilia patients with AAV liver targeting gene therapy. Recent clinical trials in patients with hemophilia B have generated variable FIX expression levels after AAV gene therapy. Some patients had less than 5% of FIX levels in the blood when low doses of AAV vector were used or they decreased to less than 5% at a late time post AAV administration.18,27 These patients may experience spontaneous bleeding episodes. If bleeding occurs, immediate administration of glucocorticoids could transiently increase FIX expression and would improve hemostasis and eventually reach therapeutic effects.

It has previously been demonstrated that the administration of dexamethasone increases the levels of some clotting factors, including FIX.28,29 Several clotting factors could be increased to different levels (such as factor VIII by 27%, factor XI by 6%) when short-term (5-day) dexamethasone treatment is applied.28 In this study, late administration of dexamethasone after AAV transduction generally increased FIX expression more than two-fold. It is possible that dexamethasone is not the only contributor to the effect of the increased transgene FIX levels. Other factors may also play a role in the effect of dexamethasone on transgene expression enhancement, which may be related to AAV vector transduction in the liver. Ironically, we did not find that the increased transgene expression with dexamethasone treatment was associated with the transcription level and genome copy number of the transgene in our study. Although there is a remote possibility that our observation is related to a technical concern, glucocorticoids have been widely used in clinics for their robust effects on inflammatory and immune processes.30 As is well known, glucocorticoids suppress inflammatory responses through regulating the process in multiple phases during inflammation.30 It has been found that glucocorticoids activate genes that encode for inhibitors of the TLR signaling pathway, including nuclear factor kappa B (NF-κB) activity.31,32 They also inhibit the expression of many pro-inflammatory cytokines, such as interleukin (IL)-1b, IL-6, and TNF.30 Moreover, glucocorticoids have functions to regulate the gene expression that is related to innate immunity.30,33 In this study, we found that the expression of TNF-α and IFN-β was decreased in the AAV-transduced liver of mice treated with dexamethasone, which is in agreement with previous findings.34 It is noteworthy that only a few studies have focused on the effect of glucocorticoids on IFN-β expression. One report showed that the expression of IFN-β was increased in both the cultured normal and inflammatory epithelial cells after dexamethasone treatment.35 However, another study found that glucocorticoids inhibited the IFN-β signaling in the lungs of the patients, consistent with our observations in this study.34 To extend this line of thought, it has been documented that IFN-β inhibits AAV transduction.21 Therefore, perhaps dexamethasone increases late transgene expression in the AAV-transduced liver via downregulation of the innate immune response and inhibition of pro-inflammatory cytokine production. It is important to note that no liver toxicity was observed in mice after dexamethasone was used long-term (Supplementary Fig. S1), implicating that higher transgene expression from dexamethasone treatment is not induced by liver toxicities.

AAV8, AAV5 and 6 have successfully been used in clinical trials with hemophilia B.36,37 However, no effect of glucocorticoids on transgene expression was observed in the mice treated with AAV5 vectors encoding human factor VIII (AAV5/hFVIII). Administration of prednisolone did not change the levels of hFVIII in the blood or the liver at the levels of DNA, RNA, or protein, or activity in the C57BL/6 mice.38 Recently, the same group reported that the administration of prednisolone before AAV treatment increased transgene expression,39 which is consistent with our studies.22 The timings for glucocorticoids treatment and detection of the transgene expression are important parameters on AAV transduction enhancement from the administration of glucocorticoids.

It is noteworthy that the effect of dexamethasone on AAV transduction enhancement was not observed in hemophilia B dogs. Although different transgenes were used, human FIX for the mouse study and canine FIX for the dog experiment, the encoded FIX transgenes were driven by the same TTR promoters in both mice and dogs. Thus, the inability to obtain increased transgene expression in both models suggests that the increased transgene expression from dexamethasone treatment in mice is not a direct result from the effect of dexamethasone on the vector promoter. In addition, no effect of dexamethasone on transgene expression was also observed in our in vitro study when dexamethasone was added at an early time (1 day) post AAV transduction in Huh 7 cells (Supplementary Fig. S2). We speculate that dexamethasone may use a different mechanism to impact transgene expression in AAV-transduced cells in different species. It has been reported that glucocorticoids increase isoprenaline-stimulated respiration UCP-1 expression in humans but decrease its expression in rodents,40 which may suggest that the glucocorticoids regulate some gene in a species-specific manner. Therefore, the different effects from dexamethasone treatment in the mice and the dogs might be related to other genes that have different functions in different species. This may also explain the various effects of glucocorticoids in different patients after AAV liver targeting. In addition, the metabolism and the pharmacokinetics of dexamethasone in different species may also contribute to the effect we observed from dexamethasone in mice versus dogs. In addition, metabolite profiles vary in different species and sexes.41 Although we do not know the pharmacokinetics of dexamethasone in dogs, it has been documented that the clearance of dexamethasone exhibits moderate variability, and distribution parameters are largely conserved across most species.42

Glucocorticoids have routinely been used to prevent liver damage in hemophilia patients receiving AAV liver targeting gene therapy. Initially, the administration of prednisolone improved liver function, blocked the further decline of transgene expression, and restored the transgene expression to the previous level.16,43 Recently, glucocorticoids have also been used to protect the liver in patients with muscular diseases and neurological disorders as a standard care when AAV vectors are systemically administered.44 It should be noted that the results in this study using dexamethasone in mice may not be identical to that with prednisolone in AAV clinical trials, although both dexamethasone and prednisolone are commonly used in clinics for anti-inflammation. These two drugs have some difference. For example, dexamethasone is five to six times as potent as prednisolone, and it has a long-acting half-life of up to 72 h, compared with prednisolone, which has a half-life of 12 to 36 h. Rationally using glucocorticoids in clinical trials is a challenge. Long-term application of glucocorticoids may cause many kinds of toxicity. In the future, it is imperative to figure out when and how long glucocorticoids as well as which glucocorticoid drug should be administered to exert the best liver protection with the least complications.

In summary, pretreatment of dexamethasone increased transgene FIX expression in mice with AAV-targeted liver gene therapy. Moreover, the transient transgene enhancement was achieved in mice when dexamethasone was administered at later time points post-AAV administration. The increased transgene expression was independent of the timing and duration of administration post AAV injection, as well as repeated administrations. The enhancement of transgene expression from dexamethasone treatment after AAV transduction may involve the inhibition of the innate immune response and pro-inflammatory cytokine expression in the AAV-transduced liver of mice. These results provided valuable information to design an effective approach for application of dexamethasone in future clinical studies with AAV vector liver targeted gene therapy, such as short-term and intermittent administration of steroids. This may decrease the toxicities from long-term application of steroids and also avoid liver damage from high doses of AAV vector seen in clinical trials.

Supplementary Material

Supplemental data
Suppl_TableS2.docx (14.3KB, docx)
Supplemental data
Suppl_TableS1.docx (12.8KB, docx)
Supplemental data
Suppl_FigureS1.docx (22.1KB, docx)
Supplemental data
Suppl_FigureS2.docx (27.9KB, docx)

ACKNOWLEDGMENTS

The authors acknowledge the UNC Biomedical Research Imaging Center (BRIC) and Small Animal Imaging (SAI) facility for assistance with mouse imaging and UNC Histology Research Core Facility for histological services.

AUTHOR DISCLOSURE

C.L. is a cofounder of Bedrock Therapeutics, Nabgen and GeneVentiv, Inc., He has licensed patents by UNC and has received royalties from these startups and Asklepios Biopharmaceutical. R.J.S. is the founder and a shareholder at Asklepios BioPharmaceutical. He holds patents that have been licensed by the UNC to Asklepios Biopharmaceutical, for which he receives royalties. He has consulted for Baxter Health care and has received payment for speaking.

FUNDING INFORMATION

This work was supported by the National Institutes of Health Grants R01AI117408 and R01HL151348 (to C.L. and R.J.S.), R01HL144661 (to C.L. and T.C.N.), P30-CA016086-35-37, N01_75N92019D00041 (to T.C.N.), and U54-CA151652-01-04 (to the BRIC SAI facility).

SUPPLEMENTARY MATERIAL

Supplementary Table S1

Supplementary Table S2

Supplementary Figure S1

Supplementary Figure S2

REFERENCES

  • 1. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 2020;21:255–272. [DOI] [PubMed] [Google Scholar]
  • 2. Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 2011;12:341–355. [DOI] [PubMed] [Google Scholar]
  • 3. Duan D. Systemic delivery of adeno-associated viral vectors. Curr Opin Virol 2016;21:16–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Pipe S, Leebeek FWG, Ferreira V, et al. Clinical considerations for capsid choice in the development of liver-targeted AAV-based gene transfer. Mol Ther Methods Clin Dev 2019;15:170–178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Berns KI, Srivastava A. Next generation of adeno-associated virus vectors for gene therapy for human liver diseases. Gastroenterol Clin North Am 2019;48:319–330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Chai Z, Zhang X, Dobbins AL, et al. Chimeric capsid proteins impact transduction efficiency of haploid adeno-associated virus vectors. Viruses 2019;11:1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Smalley E. First AAV gene therapy poised for landmark approval. Nat Biotechnol 2017;35:998. [DOI] [PubMed] [Google Scholar]
  • 8. Gordillo M, Evans T, Gouon-Evans V. Orchestrating liver development. Development 2015;142:2094–2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Kattenhorn LM, Tipper CH, Stoica L, et al. Adeno-associated virus gene therapy for liver disease. Hum Gene Ther 2016;27:947–961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Fagiuoli S, Daina E, D'Antiga L, et al. Monogenic diseases that can be cured by liver transplantation. J Hepatol 2013;59:595–612. [DOI] [PubMed] [Google Scholar]
  • 11. Baruteau J, Waddington SN, Alexander IE, et al. Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects. J Inherit Metab Dis 2017;40:497–517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. High KA. Gene therapy for hemophilia: the clot thickens. Hum Gene Ther 2014;25:915–922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Keeler GD, Markusic DM, Hoffman BE. Liver induced transgene tolerance with AAV vectors. Cell Immunol 2019;342:103728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Zhao B, Zhang HY, Xie GJ, et al. Evaluation of the efficacy of steroid therapy on acute liver failure. Exp Ther Med 2016;12:3121–3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Nathwani AC, Rosales C, McIntosh J, et al. Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins. Mol Ther 2011;19:876–885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Nathwani AC, Reiss UM, Tuddenham EGD, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014;371:1994–2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood 2019;133:407–414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. High KA, Anguela XM. Adeno-associated viral vectors for the treatment of hemophilia. Hum Mol Genet 2016;25:R36–R41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Pasi KJ, Rangarajan S, Mitchell N, et al. Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A. N Engl J Med 2020;382:29–40. [DOI] [PubMed] [Google Scholar]
  • 20. Manno CS, Arruda VR, Pierce GF, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006;12:342–347. [DOI] [PubMed] [Google Scholar]
  • 21. Shao W, Earley LF, Chai Z, et al. Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction. JCI Insight 2018;3:e120474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Chai Z, Zhang X, Dobbins AL, et al. Optimization of dexamethasone administration for maintaining global transduction efficacy of adeno-associated virus serotype 9. Hum Gene Ther 2019;30:829–840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Chai Z, Sun JJ, Rigsbee KM, et al. Application of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody evasion. J Control Release 2017;262:348–356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Du LM, Nurden P, Nurden AT, et al. Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A. Nat Commun 2013;4:2773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Becker DE. Basic and clinical pharmacology of glucocorticosteroids. Anesth Prog 2013;60:25–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Barnes PJ. Glucocorticosteroids: current and future directions. Br J Pharmacol 2011;163:29–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Nathwani AC, Tuddenham EGD, Rangarajan S, et al. Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 2011;365:2357–2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Brotman DJ, Girod JP, Posch A, et al. Effects of short-term glucocorticoids on hemostatic factors in healthy volunteers. Thromb Res 2006;118:247–252. [DOI] [PubMed] [Google Scholar]
  • 29. Ueda N, Kawaguchi S, Niinomi Y, et al. Effect of corticosteroids on coagulation factors in children with nephrotic syndrome. Pediatr Nephrol 1987;1:286–289. [DOI] [PubMed] [Google Scholar]
  • 30. Cain DW, Cidlowski JA. Immune regulation by glucocorticoids. Nat Rev Immunol 2017;17:233–247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Miyata M, Lee JY, Susuki-Miyata S, et al. Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M. Nat Commun 2015;6:6062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Beaulieu E, Morand EF. Role of GILZ in immune regulation, glucocorticoid actions and rheumatoid arthritis. Nat Rev Rheumatol 2011;7:340–348. [DOI] [PubMed] [Google Scholar]
  • 33. Galon J, Franchimont D, Hiroi N, et al. Gene profiling reveals unknown enhancing and suppressive actions of glucocorticoids on immune cells. FASEB J 2002;16:61–71. [DOI] [PubMed] [Google Scholar]
  • 34. Jalkanen J, Pettilä V, Huttunen T, et al. Glucocorticoids inhibit type I IFN beta signaling and the upregulation of CD73 in human lung. Intensive Care Med 2020;46:1937–1940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Hwang JW, Lee KJ, Choi IH, et al. Decreased expression of type I (IFN-β) and type III (IFN-λ) interferons and interferon-stimulated genes in patients with chronic rhinosinusitis with and without nasal polyps. J Allergy Clin Immunol 2019;144:1551–1565.e2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Majowicz A, Nijmeijer B, Lampen MH, et al. Therapeutic hFIX activity achieved after single AAV5-hFIX treatment in hemophilia B patients and NHPs with pre-existing Anti-AAV5 NABs. Mol Ther Methods Clin Dev 2019;14:27–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Spronck EA, Liu YP, Lubelski J, et al. Enhanced factor IX activity following administration of AAV5-R338L “Padua” factor IX versus AAV5 WT human factor IX in NHPs. Mol Ther Methods Clin Dev 2019;15:221–231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Zhang L, Handyside B, Murphy R, et al. Prednisolone does not regulate factor VIII expression in mice receiving AAV5-hFVIII-SQ: valoctocogene roxaparvovec. Mol Ther Methods Clin Dev 2020;17:13–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. BioMarin harmaceutical Inc., Novato, CA.. ASGCT Annual Meeting Abstracts. Mol Ther 2021;29:1–427.33308439 [Google Scholar]
  • 40. Ramage LE, Akyol M, Fletcher AM, et al. Glucocorticoids acutely increase brown adipose tissue activity in humans, revealing species-specific differences in UCP-1 regulation. Cell Metabol 2016;24:130–141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Tomlinson ES, Maggs JL, Park BK, et al. Dexamethasone metabolism in vitro: species differences. J Steroid Biochem Mol Biol 1997;62:345–352. [DOI] [PubMed] [Google Scholar]
  • 42. Song D, Jusko WJ. Across-species meta-analysis of dexamethasone pharmacokinetics utilizing allometric and scaling modeling approaches. Biopharm Drug Dispos 2021;42:191–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. George LA, Sullivan SK, Giermasz A, et al. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med 2017;377:2215–2227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Mendell JR, Moxley RT, Griggs RC, et al. Randomized, double-blind six-month trial of prednisone in Duchenne's muscular dystrophy. N Engl J Med 1989;320:1592–1597. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental data
Suppl_TableS2.docx (14.3KB, docx)
Supplemental data
Suppl_TableS1.docx (12.8KB, docx)
Supplemental data
Suppl_FigureS1.docx (22.1KB, docx)
Supplemental data
Suppl_FigureS2.docx (27.9KB, docx)

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