Enhancing AAV9-UFμDys1 Gene Therapy Efficacy Through Immunosuppression in Mice with Pre-Existing Immunity and Enabling Redosing Strategies for Duchenne Muscular Dystrophy

Abstract

Significant progress has been made in gene therapy for Duchenne muscular dystrophy (DMD), a severe genetic disorder primarily affecting pediatric patients. However, the immune responses triggered by high-dose systemic delivery of adeno-associated virus (AAV) vectors remain a major challenge. These responses include the generation of long-lasting anti-capsid antibodies and potential immunity against the therapeutic transgene, rendering gene therapy ineffective. In addition, pre-existing anti-AAV antibodies exclude patients from eligibility for treatment. To address these limitations, we have developed an immunosuppression (IMS) strategy aimed at mitigating immune responses to the AAV capsid while enhancing microdystrophin expression. Using an optimized expression cassette (AAV9-UFµDys1) for sustained microdystrophin expression in striated muscle and heart, we observed a 40% improvement in muscle force compared with animals receiving a GFP-encoding control AAV9 vector. In mdx mice, a single-dose IMS regimen significantly increased microdystrophin expression in cardiac and skeletal tissues and repeat dosing further enhanced expression, an effect not observed in non-IMS-treated mdx mice. To model pre-existing immunity, we immune-challenged wild-type mice with empty AAV9 capsids and tracked antibody responses over time. The IMS regimen effectively reduced total anti-AAV antibody levels and increased microdystrophin expression in UFµDys1-treated mice. These findings highlight the potential of IMS to minimize immune barriers, facilitate repeat AAV administration, and expand the therapeutic window for DMD gene therapy. Our results support the further development of AAV-mediated approaches using either microdystrophin-expressing vectors or next-generation systems delivering full-length or near-full-length dystrophin.

Keywords

AAV gene therapy preexisting antibodies DMD redosing AAV immune response dystrophin

INTRODUCTION

Despite remarkable progress in systemically administered adeno-associated virus (AAV) gene therapy, a pivotal challenge of host immune responses directed against the viral vector capsid and therapeutic transgene persists. Heightened immune responses raise concerns regarding the safety and long-term efficacy of gene therapy. Insights from our preclinical investigations emphasize that previous preexisting immunity can culminate in severe infusion reactions and a concomitant loss of therapeutic impact. Considering these inherent limitations, ongoing clinical trials using AAV-mediated gene therapy have been compelled to adopt a strategy of single-vector exposure while concurrently excluding subjects possessing preexisting immunity to AAV. In humans, it has been consistently shown that the administration of AAV vectors in the dose range required for regional or systemic exposure leads to high and sustained anti-AAV antibodies.^1–3 In the current report, we establish the basis for effectively regulating the host immune response against the vector capsid and the transgene product.

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder characterized by the progressive degeneration of muscle tissue, primarily affecting young boys.⁴ It is caused by mutations in the dystrophin gene, leading to the absence or dysfunction of the dystrophin protein, which plays a crucial role in maintaining muscle integrity.⁵ Gene therapy for DMD entails the systemic administration of a therapeutic dystrophin transgene via AAV vectors. Besides the potential immunogenicity of the viral vector, which may trigger immune responses in patients,⁶ the use of AAV in DMD gene therapy is also limited by their restricted capacity to carry genetic material, which is approximately 4.7 kb. This limitation presents a significant challenge in the treatment of DMD, where the dystrophin gene exceeds 2.2 million base pairs in size. To address this issue, truncated cDNA constructs (microdystrophin) have been developed.⁷ These shortened constructs remove large portions of the dystrophin sequence while retaining critical domains such as hinges, repeats, and structural elements believed to be vital for the protein’s overall functionality. Studies involving dystrophic animal models have demonstrated that the administration of microdystrophin constructs is well-tolerated, and that the resulting microdystrophin proteins maintain significant functional capacity within striated muscles. Furthermore, they have been shown to enhance the functional attributes in animal models.^8–10 SRP-9001/ELEVIDYS, a microdystrophin gene therapy, has recently been approved by the FDA for DMD patients.^11–13 In this study, we utilize a codon-optimized human microdystrophin construct referred to as UFµDys1, and investigate its expression in mdx mice, a DMD mouse model, comparing our findings with those of previously successful microdystrophin 5 (µDys5),^10,14 used in DMD clinical trials (NCT03368742). AAV gene therapy with µDys5 has several limitations, including the following: incomplete muscle correction, potential for cardiac complications due to overexpression, concerns about durability as satellite cells do not express microdystrophin, and the possibility of muscle damage leading to loss of microdystrophin-expressing cells seen in mdx mice.¹⁵ The spectrin-like repeat R2 domain enables microdystrophin to localize in muscle sarcolemma rather than being distributed in the cytosolic compartment.¹⁶ The µDys5 construct lacks the R2 domain, which is present in UFµDys1. The rod domain, R2, is essential for maintaining the structural integrity of dystrophin, allowing it to act as a linker between the sarcolemma and the intracellular cytoskeleton, resisting mechanical stress during muscle contraction.^16,17 The R2 domain was reported to harbor seven mutations in DMD and one mutation in intermediate muscular dystrophy.¹⁸

Antibody generation resulting from early-life environmental exposure to AAV is a common phenomenon, and can significantly impact the applicability of AAV as a gene therapy vector.¹⁹ When exposed to antigens through natural or therapeutic means, naive B cells undergo differentiation, forming memory B cells and antibody-secreting plasma cells. Long-lasting IgG and IgM plasma cells can persist indefinitely within the bone marrow and spleen.^20,21 Our strategy to mitigate the immune response in AAV-mediated gene therapy involves pharmacological modulation of the humoral immune system using bortezomib and daratumumab, either alone or in combination with sirolimus and anti-CD20 antibody. Our model combines immune agents to deplete long-lived plasma cells and target B cells, potentially reducing anti-AAV antibodies for gene therapy in patients with moderately elevated levels. Anti-CD20 antibody, a monoclonal antibody, targets B cells by binding to CD20. It functions by inducing cell lysis through complement activation, engaging in antibody-dependent cell-mediated cytotoxicity, or by directly triggering apoptosis.²² Sirolimus, an inhibitor of B cell proliferation,²³ demonstrates a selective capacity to enhance the survival and proliferation of regulatory T cells while facilitating programmed cell death of activated effector T lymphocytes.^24,25 Sirolimus has been found to enhance the population of regulatory B cells and regulatory T cells in liver transplant patients. This immunosuppressive drug promotes immune tolerance, potentially reducing the risk of rejection and improving transplant outcomes.²⁶ In gene therapy, nonclinical studies have demonstrated the effectiveness of sirolimus as an immunomodulator.^27,28 Sirolimus is complementary to the effect of anti-CD20 as an immunomodulatory agent.²⁹ Preclinical animal studies and clinical studies support the safety and efficacy of immunomodulation for repeat AAV administration, preventing antibody formation against the transgene and capsid.³⁰ Pompe disease, a rare genetic disorder, is being targeted for gene therapy to correct enzyme deficiencies.³¹ Clinical evaluation in adult patients afflicted by Pompe disease corroborates preclinical outcomes showing that the administration of an anti-BAFF therapy alongside recombinant human GAA significantly reduced antidrug antibodies in enzyme replacement therapy, substantiating the potential of pharmacological immunomodulation for immune response attenuation and facilitating the feasibility of vector readministration.³²

Additional agents have been considered to enhance the effect of anti-CD20 therapy. Bortezomib inhibits proteasomes and can eliminate long-lived plasma cells, aiding in managing sustained antibody titers in enzyme replacement therapy for Pompe disease.³³ Daratumumab targets CD38-expressing cells, including plasma cells and immune cells, showing efficacy in vitro³⁴ and in preclinical studies,^35,36 and has received FDA approval for multiple myeloma.³⁷

Repeated systemic dosing may play an important role in DMD gene therapy to achieve a greater level of dystrophin-positive fibers, enhance safety of initial exposure, and to preserve durability of the therapeutic gene. The concept of repeat systemic dosing addresses these considerations and enables safe and effective repeat vector administration, extending the therapeutic window and refining the treatment of various conditions, especially DMD.^37,38 Repeated dosing is also a critical consideration in Pompe gene therapy, as therapeutic effects may wane over time due to factors such as immune responses and declining functional gene copies. Our previously published work confirms the safety of single and repeat administration of the rAAV9-DES-hGAA vector for Pompe disease, combined with an immunosuppressive regimen preventing antibody formation against the GAA transgene and rAAV9 capsid.³⁹

Here, we have developed and optimized alternative strategies for immunosuppression (IMS) to prevent immune responses against the AAV capsid and effectively manage preexisting immunity to AAV while enabling readministration of gene therapy for DMD.

MATERIALS AND METHODS

AAV vector design, cells, plasmids, viruses, and transfection

The UFµDys1 cassette consists of a creatine kinase 8 (CK8) promoter, and a 3.7 kb microdystrophin transgene derived from key domains of full-length dystrophin was packaged into the AAV9 vector (Fig. 1A). We used the human microdystrophin µDys5 as a positive control¹⁰ (Fig. 1B). The UFµDys1 differs from µDys5 in that it includes the R2 domain and excludes R23. Viral vectors (AAV9-CMV-GFP, AAV9-CK8-µDys5, AAV9-CK8-UFµDys1) were produced through a double-transfection protocol at the Powell Gene Therapy Center, University of Florida,^40,41 and purity was assessed by sodium dodecyl sulfate (SDS)–polyacrylamide gel (PAGE) (Supplementary Fig. S1).

Figure 1.

In vitro UFµDys1 protein expression in HEK293 cells and microdystrophin schematic comparisons. (A) Schematic diagram showing the domains of UFµDys1 (B) Western blot of lysate from HEK293 cells transfected with CMV-UFµDys. The Western blot data show full-length human dystrophin from human cardiac biopsy samples in control patients (lanes 1–2), UFµDys1 after transfection in HEK293 cells (lane 3), and negative control in nontransfected cells (lane 4). Vinculin was used as a protein loading control. (C) Key features of clinically significant miniaturized dystrophin constructs. In the top two panels, muscle (Pfizer)- and heart (Sarepta Therapeutics)-specific promoters, depicted by yellow arrows, are used in these therapeutic constructs, ensuring preferential transgene expression in these tissues. The transgene within all constructs includes coding sequences for clinically relevant and functional dystrophin domains. The choice of adeno-associated virus (AAV) serotype is guided by tissue tropism, focusing on muscle and heart tissues. The UFµDys1 construct differs from µDys5 in that it includes the R2 spectrin repeat and excludes R23.

HEK293 cells (ATCC, Manassas, VA) were cultured in DMEM with 10% fetal bovine serum (FBS) and penicillin–streptomycin antibiotics for the electroporation transfection process. In vitro experiments in cultured HEK293 cells confirmed microdystrophin expression after transfection with 5 µg of CMV-UFµDys1 plasmid. Heart tissue control biopsy samples were used for comparing full-length human dystrophin and microdystrophin (Fig. 1B).

Western blots

Proteins were extracted using RIPA buffer and quantified using BCA assay (Sigma). Fifty micrograms of protein were loaded on a 4–12% gradient SDS-PAGE (Life Technologies) and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% milk/TBST, probed with dystrophin (1:100, DHSB), vinculin and GAPDH antibodies (1:1,000, Cell Signaling Technology), and detected using HRP-conjugated secondary antibodies and chemiluminescence (Millipore). Known amount of recombinant µDys1 protein was used to calculate the amount of µDys in animal tissues.

Animals

Neonatal C57BL/10 wild-type study

Four groups (one litter per group) of male and female C57BL/10 mice received AAV9-UFµDys1 on postnatal day 1 (P1) via intraperitoneal injection to observe the initial expression of the vectors compared with positive controls such as AAV44.9-µDys5 and AAV9-µDys5 (Supplementary Fig. S2).

Mdx mice study

C57BL/10ScSn-Dmdmdx/J male mice (stock #001801) were sourced from Jackson Laboratory, USA, and acclimatized for 7 days. Cages housed up to five mice. Animals were handled per AGADA Biosciences Inc, Nova Scotia, Institutional Animal Care and Use Committee (IACUC) guidelines for efficacy studies. The mdx mice were injected intravenously with a dose of 1 × 10¹⁴ vg/kg AAV9-UFµDys1 as indicated in Fig. 2A for exhaustion assay, in vitro muscle force assay, and histological analysis.

Figure 2.

Systemic UFµDys1 delivery restores dystrophin expression in the striated musculature of mdx mice and improves whole-body disease indices. (A) C57BL/10ScSn-Dmdmdx/J (mdx) mice study design. (B) Viral vector titers. (C) Average distance ran by mdx mice receiving AAV9-UFµDys1, AAV9-µDys5, or a nonrelevant AAV9-GFP construct in an exhaustion assay. We observed that UFµDys1-injected mice (N = 5) performed similar to the µDys5-injected positive control (N = 6) as seen by the normalized distance covered by mdx mice. (D, E) In vivo measurements of extensor digitorum longus (EDL) maximal force and specific force in microdystrophin-injected experimental mice show higher muscular force than negative control GFP-injected mdx mice. The statistical analysis compared mdx AAV9-GFP (negative control) and other microdystrophin groups using one-way ANOVA followed by multiple comparisons between each treatment group using the Sidak multiple comparison method (mean ± SEM; p values are shown). (F) The top panel shows representative images of dystrophin immunofluorescent staining in frozen heart tissue sections from each treatment group. Dystrophin-positive areas are indicated in red. The bottom panel shows representative images of Picro-Sirius Red staining in frozen heart tissue sections from each treatment group, showing collagen-positive areas in red. (G) Quantification of dystrophin-positive area in heart tissues on each treatment group. Analysis was performed using one-way ANOVA with multiple comparisons using the Sidak method to compare conditions. Data represented as mean ± SEM, p values shown. (H) Quantification of collagen-positive area in heart tissues on each treatment group. Analysis was performed using one-way ANOVA with multiple comparisons using the Sidak method to compare conditions.

Exhaustion assay

At week 6 post-AAV injection, a treadmill with four lanes (Columbus Instruments) was used for an exhaustion assay. Each mouse ran for 5 min at 5 m/min for acclimatization. After acclimatization, the speed was increased to 1 m/min for 5 min. Exhaustion was defined as the mouse being unable to continue running or resting for 30 s despite nudges. Each mouse underwent the assay three times every other day, recording stop times, average running distance (m), and calculating normalized distance (m/kg).

In vitro muscle force

In vitro contractile properties of the right extensor digitorum longus (EDL) muscle were assessed in anesthetized mice at week 5 post-AAV injection/gene therapy. The EDL muscles, immersed in oxygenated Ringer’s solution at 25°C, were stimulated to measure maximal force at 10 mN resting force. Stimulation frequency was increased until a plateau (∼250Hz) was reached, representing maximum force (mN). Muscle cross-sectional area was calculated from mass, length, and density (1.056 g/mL), and muscle-specific force (kN/m²) was determined based on cross-sectional area.

Histology

Fresh frozen heart tissues (n = 8/group) were sectioned at 10 μm thickness. Dystrophin levels and collagen were analyzed through immunofluorescence (IF) and Picro Sirius Red (PSR) staining, respectively. The IF staining was performed using antibody recognizing exons 10/11 of dystrophin (MANEX 1011B) (1:50 dilution) and DAPI counterstaining. PSR staining highlighted collagen in red. Image analysis in ImageJ included calculating the percent area of dystrophin-positive fibers and the percentage of fibrosis by measuring the red collagen area relative to the total section area, represented in 3–7 snaps per image and average was considered. Analysis was done with ImageScope and ImageJ software. Exhaustion assay, in vitro muscle force assay, and histological analysis were performed at AGADA Biosciences Inc, Nova Scotia.

IMS in mdx mice

We used C57BL/10ScSn-Dmdmdx/J male mice (stock #001801) from Jackson Laboratory, USA, and acclimatized them for 7 days. Animals were handled as per University of Florida IACUC guidelines for immunomodulation studies. The Mdx mice were then treated with anti-CD20 (3.3 mg/kg) [clone 18B12, Biogen] at two weekly doses before the AAV dose, with maintenance doses of 3.3 mg/kg at 10 and 14 weeks of age, along with sirolimus (five weekly 1 mg/kg doses via oral gavage) [Amneal] (Supplementary Fig. S4). Thirty minutes before the administration of anti-CD20, 50 mg/mL diphenhydramine was administered to the animals to combat any cytokine storm.

Mimicking of preexisting immunity using empty AAV9 capsids and reducing antibody response by various methods of IMS in wild-type mice

Five-week-old adult wild-type (C57BL/10) mice received a 50 μg dose of empty AAV9 capsids (Fig. 5A, B) injected intravenously to simulate preexisting immunity. They were then treated with anti-CD20 (3.3 mg/kg) [clone 18B12, Biogen] at day 70, along with sirolimus (1 mg/kg daily) [Amneal]. Bortezomib (0.75 mg/kg twice weekly) [SelleckChem #S1013] and daratumumab (16 mg/kg weekly) [SelleckChem #A2027] were also administered either alone or in combination with anti-CD20 antibody and sirolimus. Diphenhydramine (50 mg/mL) was administered to these animals to combat any cytokine storm before the anti-CD20 antibody dose.

Evaluation of preexisting immunity simulation and IMS effects after UFµDys1 administration in wild-type mice

A similar pattern of IMS regimen was followed as previously stated. At day 70, AAV9-UFµDys1 vector was administered (Fig. 6A, B). The IMS regimen for these animals continued until day 100. This protocol was designed to evaluate the effects of simulated preexisting immunity and various IMS methods on µDys administration in wild-type mice. The timeline of interventions was structured to enable the establishment of preexisting immunity, followed by IMS and the subsequent gene therapy administration.

Anti-AAV9 ELISA

Ninety-six-well microplates (Immulon 4HBX, Thermo Fisher) were coated with 1.2 × 10¹¹ AAV9 particles in 0.5M sodium bicarbonate (pH 8.4) and incubated at 4°C overnight. After washing with 1× PBST, plates were blocked with 10% heat-inactivated FBS in 1× PBS at 37°C for 2 h. Serum samples were serially diluted (1:10 to 1:102,400) in a low-binding plate using blocking buffer. Sample concentration (U/mL) was determined using a four-parameter logistic standard curve from up to three dilution points within the linear range.²⁸

Droplet digital polymerase chain reaction methods

Viral vector titer:

The quantification of vector genomes per ml (vg/mL) was designed focusing on the codon-optimized human UFµDys1 gene as the target while unable to bind to native murine dystrophin.

Primer sequences:

Primer 1: 5′-TGCTGCTTGCCGAACTTG-3′

Primer 2: 5′-CGAGGACGTGCAGAAGAAAC-3′

Probe: 5′-/56-FAM/TCACCAAAT/ZEN/GGGTCAACGCCCA/3IABkFQ/-3′

Bio-Rad droplet digital polymerase chain reaction (ddPCR) supermix (without dUTP) was used, and primers were added at a concentration of 540 nm, with the probe at 150 nm. Titer calculations were performed using QX-200 copy/µL values, incorporating all dilution factors, and taking an average of three replicates for each dilution (Fig. 2B).

RNA, cDNA isolation, and analysis:

The expression of UFµDys1 was assessed using RNA extracted from mouse tissue, following an optimized RT-ddPCR method. Mouse RPP30 ribonuclease was used as a reference target. For RNA extraction, tissue weight was recorded, and tissues were lysed using the Invitrogen PureLink RNA Mini Kit. Subsequently, cDNA was synthesized using the High Capacity Reverse Transcription Kit with Multiscribe and an RNASE inhibitor. The resulting cDNA copies were quantified for µDys copy numbers.

Human µDys-specific cDNA primers and probe:

Primer 1: 5′-TGCTGCTTGCCGAACTTG-3′

Primer 2: 5′-CGAGGACGTGCAGAAGAAAC-3′

Probe: 5′-/56-FAM/TCACCAAAT/ZEN/GGGTCAACGCCCA/3IABkFQ/-3′

Murine RPP30 cDNA primers and probe:

Primer 1: 5′-GAGGGCATTGGAGATTGTG-3′

Primer 2: 5′-CCTGGGCTTTGAACTTGTC-3′

Probe: 5′-/5HEX/TGGTCCTGC/ZEN/TATCAGAGATGCAACG/31ABkFQ/-3′

Statistical analyses

Quantitative data are displayed as individual data points as mean ± SEM. All statistical analyses were performed using Prism v.10.1 statistical software (GraphPad). The correct statistical test for each analysis was determined by first assessing the normality and variance of the data. One-way and two-way ANOVAs were performed to determine the statistical significance. Dunnett’s and Sidak’s multiple-comparison post hoc tests were used to compare data between the groups. A p value of ≤0.05 was considered significant.

Study approval

Plasmid constructs were generated in accordance with the Environmental Health and Biosafety guidelines at the University of Florida. Mouse husbandry and breeding were carried out under the supervision of Animal Care Services at the University of Florida, following protocols approved by the IACUC.

RESULTS

Human µDys successfully expresses in mammalian HEK293 cells after transfection

To confirm expression of µDys in vitro, we transfected HEK293 cells with CMV-µDys, and performed Western blot on the cell lysate using the anti-dystrophin antibody (MANEXA44A). Human cardiac biopsy tissue lysates were used as a control, confirming that antibody can detect both full-length dystrophin and µDys proteins (Fig. 1B). The µDys construct, as well as other microdystrophin constructs, including the functional domains comparison between human µDys5 and human UFµDys1, are shown in Fig. 1C. Spectrin-like domain R2 was included in UFµDys1 and R23 was excluded. Both expression cassettes included the R16-R17 nNOS binding domain.

Administration of AAV9-UFµDys1 vector to mdx mice improves exhaustion recovery and muscle function

To assess AAV9-UFµDys1 gene therapy vector efficacy in young adult C57BL/10ScSn DMDmdx/J male mice, 5-week-old mice were divided into three treatment groups, as indicated in Fig. 2A, and received intravenous (IV) administration of the different AAV9 constructs at 1 × 10¹⁴ vg/kg (Fig. 2A, B). A codon-optimized version of microdystrophin µDys5 was used as a positive control. The µDys5 construct has been reported to show significant improvements in muscle pathology and function after gene therapy administration in mdx mice.¹⁴ Functional outcome of AAV9-UFµDys1, AAV9-µDys5, and AAV9-GFP vectors was evaluated through weekly body weights, treadmill exhaustion, in vitro muscle force measurements, and in situ nerve-stimulated contraction tests. Mice receiving the UFµDys1 vector showed a statistically significant recovery at week 4 posttreatment (8 weeks of age) in treadmill exhaustion of 94% (p = 0.0148) (Fig. 2C), a 40% improvement in muscle function in the tibialis anterior (p = 0.0177) (Fig. 2D), and a 32% recovery of in vitro muscle function (p = 0.0182) (Fig. 2E) in comparison with the GFP negative control. No significant differences in these parameters were observed between the positive control (µDys5) and the test article (UFµDys1)-treated animals. No significant changes in tissue weight were observed (Supplementary Fig. S3). One mouse in the UFµDys1 group was euthanized due to hydrocephalus.

Injection with AAV9-UFµDys1 leads to an increase in dystrophin-positive muscle fibers in mdx mice

Dystrophin and collagen expression patterns were analyzed in frozen heart tissue sections collected week 5 posttreatment (9 weeks of age) using IF and PSR staining. The systemic administration of µDys5 and UFµDys1 resulted in 99% dystrophin-positive myofibers compared with mdx mice receiving an irrelevant GFP construct (Fig. 2F, G). The IF staining with exon 10/11 dystrophin antibody and DAPI counterstaining revealed microdystrophin localization present uniformly around membranes in a similar pattern as wild-type dystrophin⁴² due to the strong transgenic overexpression of the microdystrophin transgene in skeletal muscle. The PSR staining showed no significant differences in collagen content among the experimental groups and compared with AAV9-GFP controls because the onset of collagen infiltration in the heart starts around 3 months of age in mdx mice⁴³ (Fig. 2H).

IMS with anti-CD20 and sirolimus enhances repeat UFµDys1 administration in mdx mice

After confirming UFµDys1 expression and efficacy in mdx mice, we sought to identify, develop, and optimize a safe and effective IMS strategy for repeated administration of AAV-mediated gene therapy for the treatment of DMD. Twenty-seven C57BL/10ScSn-Dmdmdx/J male mice were divided into four treatment groups, receiving one high dose (1 × 10¹⁴ vg/kg) of AAV-UFµDys1 on day 0 or two low doses (6.6 × 10¹³ vg/kg) on day 0 and day 21, with or without IMS (Fig. 3A, B). The IMS regimen consisted of treatment with anti-CD20 antibody (3.3 mg/kg), including an induction regimen at weeks 1 and 2 before AAV injection and a maintenance regimen until the end of the study around day 100. Daily administration of sirolimus (1 mg/kg) started 3 days before the first AAV dose and was maintained through week 8. Serum samples were collected on day 0, day 35, and at day 70. Tissue samples (heart and skeletal muscle) were collected at endpoint, and dystrophin expression was quantified using densitometric analysis of microdystrophin protein (Fig. 3C). Western blot analysis confirmed high-level UFµDys1 expression in both skeletal muscle and heart tissues. Heart tissues from mice treated with a single high dose of AAV-UFµDys1 and IMS exhibited a 12.6-fold increase (p = 0.0173) in microdystrophin expression compared with mice receiving the single high dose without IMS treatment. Notably, we observed a 3.5-fold increase in cardiac protein expression (p ≤ 0.0001) when administering two low doses with IMS compared with a single high dose with IMS. Furthermore, the two low doses with IMS showed a twofold increase (p = 0.0002) in expression compared with the two low doses without IMS treatment (Figs. 3C and 4B). These results demonstrate the significant impact of dosing strategy and IMS on microdystrophin expression levels in treated tissues. The ddPCR quantification showed that IMS resulted in higher dystrophin transcript levels in both heart and skeletal muscle (Supplementary Fig. S5A) compared with the absence of IMS. We also observed a 0.9-fold increase (nonsignificant) in microdystrophin expression in skeletal muscle in animals receiving IMS and a single high dose compared with animals receiving a single high dose and no IMS. There was a 3.7-fold significant (p = 0.0078) increase in microdystrophin expression in skeletal muscle in animals receiving IMS and two repeat low doses compared with animals receiving two repeat low doses and no IMS. There was also a nonsignificant 1.9-fold increase in microdystrophin in animals receiving two low doses with IMS compared with animals receiving a single dose with IMS (Fig. 4B). ddPCR analysis confirmed consistent µDys transcript levels (Supplementary Fig. S5A).

Figure 3.

Immunosuppression (IMS) with anti-CD20 and sirolimus decreases AAV9-specific immune response and enhances redosing of UFµDys1 in mdx mice. (A) C57BL/10ScSn-Dmdmdx/J (mdx) mice IMS study design. (B) Schematic study design for IMS regimen in mdx mice. (C) Western blot analysis of UFµDys1 expression in the heart (top) and quadriceps muscles (bottom) from mdx mice after IMS and single or repeat administration of adeno-associated virus (AAV) (5 weeks of age, at least n = 4 per group). Analysis was conducted at 18 weeks of age. µDys1 was loaded as a positive control at three different protein concentrations and GAPDH was used as a loading control.

Figure 4.

Immunosuppression (IMS) reduces anti-AAV9 capsid antibody responses and enhances microdystrophin (µDys) expression following single or repeat dosing. (A) Quantification of anti-AAV9 circulating antibodies after single or repeat systemic doses of AAV9-UFµDys1 with or without IMS in mdx mice. (B) Densitometry quantification of the microdystrophin band intensity of the Western blots was analyzed via ImageJ, and protein quantification was performed based on the known amount of µDys1 protein (mean ± SEM; p values are shown).

Anti-AAV9 ELISA showed that the IMS regimen successfully reduced anti-capsid antibody levels by 31% (p = 0.0098) after a single high dose. Animals receiving repeat low doses and IMS had a significant (p = 0.0005) decrease in antibodies of 25% compared with the group receiving repeat low doses without IMS. There was also a 30% increase in antibodies in animals treated with two repeat low doses and IMS when compared with a single high dose with IMS (p = 0.04) (Fig. 4A).

Combination IMS regimen of anti-CD20, sirolimus, and bortezomib reverses preexisting humoral and cellular immune responses to AAV vector in wild-type mice

Next, we aimed to deplete long-lived plasma cells, responsible for preexisting anti-capsid antibodies, using a combination of immunosuppressive agents in a preimmune animal model. We administered empty AAV9 capsids to 32-month-old wild-type (C57BL/10) mice to mimic the preexisting AAV antibody scenario in gene therapy trials. Anti-CD20 antibody (3.3 mg/kg), sirolimus (1 mg/kg), bortezomib (0.75 mg/kg), and daratumumab (16 mg/kg), administered individually or in combination, were used for B cell depletion (Fig. 5A, B and Supplementary Fig. S6).

Figure 5.

Immunosuppression (IMS) regimen, including anti-CD20, sirolimus, and bortezomib, reverses preexisting AAV9 immunity in WT mice. (A) Schematic for study design for IMS to lower preexisting immunity in wild-type mice. Systemic delivery of empty adeno-associated virus (AAV) capsid increases anti-AAV9 antibody titer above the baseline at day 0 (N = 5–10/group). (B) Study design to address the preexisting immunity in wild-type mice with route of administration (ROA). (C) Anti-AAV9 immunity as measured by % change in anti-AAV9 antibodies after 4 weeks of IMS (n = 5–10/group). IMS decreases circulating antibodies after administration of empty AAV9 in wild-type mice (mean ± SEM; p values shown).

Serum samples were collected at three time points: day 0, day 22, and day 70 (endpoint) to measure antibody levels and evaluate the immune depletion strategy’s effectiveness (Fig. 5A). At day 70, treatment with anti-CD20 antibody + sirolimus yielded a threefold reduction in AAV9 antibody levels compared with the no-IMS control (p = 0.012). Bortezomib treatment led to a fivefold reduction (p < 0.0001) in antibody levels, compared with control mice receiving no-IMS. Mice treated with a combination of anti-CD20 antibody, sirolimus, and bortezomib exhibited a 7.5-fold reduction (p < 0.0001) compared with no IMS (Fig. 5C). Daratumumab alone reduced total antibodies (TAbs) by fivefold (p < 0.0001), indicating no additional benefit compared with bortezomib alone, whereas combination therapy with anti-CD20 and sirolimus reduced TAbs by 3.8-fold (p < 0.0001) compared with no IMS, a much weaker response than the same combination therapy harboring bortezomib instead (Supplementary Fig. S6A, B).

Our results confirm that the combination of anti-CD20 antibody, sirolimus, and bortezomib is the most effective IMS regimen for targeted depletion of existing plasma cells in animal models. The decrease in preexisting AAV9 antibodies demonstrates the potential of this therapeutic approach to reduce persistent autoantibody production, enhancing the safety and efficacy of AAV-based gene therapies.

Identification of optimal IMS regimen to enhance dystrophin expression following therapeutic AAV-UFµDys1 administration in preimmune wild-type mice

To determine the most effective immunosuppressive regimen for elevating microdystrophin levels, we utilized a cohort of 15 adult wild-type mice that had been preimmunized and divided them into three treatment groups (Fig. 6A, B). On day 0, wild-type mice received empty AAV capsids to induce an antibody response simulating preexisting immunity. On day 22, one group received an IMS regimen consisting of anti-CD20 antibody (3.3 mg/kg) and sirolimus (1 mg/kg), and another group received anti-CD20 antibody (3.3 mg/kg), sirolimus (1 mg/kg), and bortezomib (0.75 mg/kg) (Fig. 6A). A group of mice that received no IMS served as control. On day 70, mice were treated with the therapeutic vector (AAV9-µDys) at 1 × 10¹⁴ vg/kg. The study concluded on day 100, and sera and tissue samples were collected for AAV9 ELISA and µDys quantification (Fig. 6C, E).

Figure 6.

Optimizing immunosuppression (IMS) for enhanced dystrophin expression in preimmune mice treated with adeno-associated virus (AAV)-UFµDys1. (A) Schematic of the study design outlining the preimmunization and IMS dosing regimens. (B) Summary of the treatment groups. (C) Western blot analysis of microdystrophin µDys expression in the heart of wild-type mice after IMS and administration of AAV (5 weeks of age, n = 5 per group). Analysis was conducted at 20 weeks of age. GAPDH was used as a loading control. (D) Quantification of anti-AAV9 circulating antibodies after systemic doses of AAV9-UFµDys1 with or without IMS in wild-type mice. (E) Densitometry quantification of the microdystrophin band intensity of the Western blots was analyzed via ImageJ, and protein quantification was done based on the known amount of µDys1 protein (mean ± SEM; p values shown).

Results showed a significant 12-fold increase (p = 0.028) in microdystrophin protein in mice receiving anti-CD20 antibody and sirolimus, and a significant 24-fold increase (p = 0.003) in those receiving the three-drug regimen compared with no IMS treatment (Fig. 6C, E). Quantification of transcript levels of UFµDys1 through ddPCR showed consistent results (Supplementary Fig. S5B).

Circulating anti-capsid antibody levels increased at day 100 after vector administration in mice with no IMS. We observed a reduction of the IgG anti-AAV antibody production by 54% (p ≤ 0.0001) in animals receiving the anti-CD20 antibody and sirolimus regimen when compared with animals with no IMS. We also observed a 72.5% reduction (p < 0.0001) in antibody response in animals with the three-drug regimen of IMS compared with mice receiving no IMS (Fig. 6D).

DISCUSSION

In the present study, we created a new microdystrophin (UFµDys1) and tested the ability of a combination IMS therapy with clinically approved and safe immunosuppressive drugs. Here, we demonstrate that repeat administration of a divided AAV dose along with combination of IMS can increase the expression of the transgene, compared with a single cumulative dose AAV vector or the absence of IMS. Furthermore, our data support that the combination IMS regimen can successfully reduce the TAb titer against AAV in the setting of preexisting immunity and permit transgene expression after systemic AAV delivery.

In addition to the current commercial products, several other microdystrophin gene therapy clinical trials are currently underway for the treatment of DMD. AAV9-µDys5 was reported to cause serious adverse events linked to complement activation, inflammatory response, and thrombocytopenia.⁴⁴ UFµDys1 includes the R2 domain of microdystrophin for improved sarcolemma localization and could be used at a lower total exposure due to increased effectiveness in conjunction with IMS. Our findings highlight an important consideration in the development of microdystrophin-based therapies for DMD: expression levels in tissues do not necessarily correlate with functional outcomes or are indicative of potency of a clinical candidate. The comparison between UFµDys1 and µDys5 reveals a complex relationship between microdystrophin expression and functional improvement in mdx mice. While both µDys5 and UFµDys1 presented similar expression levels, µDys5 led to a higher recovery in mdx mice in terms of average distance traveled (139% improvement compared with 94% for µDys). UFµDys1-administered mice, despite showing lower recovery in the distance traveled test, exhibited superior improvements in both hypertrophy (40% vs. 22%) and specific contractile force (32% vs. 28%) of the EDL muscle when compared with µDys5. This suggests that the relationship between microdystrophin expression and functional outcomes is not straightforward and may depend on factors beyond expression levels alone. The superior muscle physiology improvements observed with UFµDys1, despite its lower impact on distance traveled, suggest that this variant might be more effective in addressing specific aspects of muscle function. Our data highlight the need for a nuanced approach in evaluating microdystrophin candidates, considering not just expression levels but also a range of functional and physiological outcomes.¹⁵

Achieving desired microdystrophin protein levels is challenging due to the development of immune responses against the AAV capsid and the encoded transgene product during the initial vector administration.⁴⁵ To overcome this, we utilized AAV redosing strategies and an IMS regimen that target B and T cell activation to prevent adaptive immune responses during initial vector administration. This IMS regimen involves the combination of anti-CD20 antibody to deplete the majority of the B cells⁴⁶ and sirolimus to inhibit T cell activation.⁴⁷ We have systematically adapted our combination drug regimen and schedule to reduce immune responses during vector dosing and allow for vector readministration without compromising therapeutic transgene expression levels in mdx mice (Fig. 3). Use of combination therapies to combat the adverse immune reactions in mdx is challenging as the immune system is dominated by proinflammatory immune cells.⁴⁸ Immunomodulatory drugs such as VBP6, prednisolone, CTLA4-Ig, and eplerenone showed variability in combating the immune response from a single dose of microdystrophin gene therapy.⁴⁹ Our group has shown that the rituximab and sirolimus regimen has proven to be effective in Pompe disease and Canavan disease.²⁷ Salabarria et al. evaluated patients receiving AAV9 for multiple conditions, including spinal muscular atrophy, GM1 gangliosidosis, DMD, and Danon disease.² In this report, patients receiving rituximab and sirolimus showed lower total anti-capsid antibodies than patients treated with corticosteroids alone. The preconditioning IMS regimen reduced thrombotic microangiopathy in patients.^2,50 It is intriguing that vector readministration after anti-CD20 antibody and sirolimus treatment at a timepoint when the immune system was not yet fully reconstituted modestly enhanced efficacy compared with controls with no IMS.² Although the underlying mechanism remains to be uncovered, this phenomenon could be exploited to achieve the desired clinical effect at low vector doses.

We used TAb assays to detect neutralizing and non-neutralizing anti-AAV antibodies. The assay can detect all anti-AAV antibody isotypes, including those of low avidity.²¹ Ultimately, the choice between TAb and neutralizing antibody (NAb) assays depends on the specific requirements of the gene therapy program and the research or clinical questions being addressed. Preexisting anti-AAV antibodies are a common cause for AAV gene therapy inegibility,⁵¹ and FDA-approved AAV-based gene therapies, Zolgensma, Elevidys, ROCTAVIAN eligibility, depend on TAb titers.⁵² Furthermore, following systemic AAV vector infusion, patients typically develop high-titer, multiserotype cross-reactive AAV NAbs that can prevent repeat AAV vector infusions from being effective. These antibodies can persist for up to 15 years.⁵³ We used wild-type mice and immune challenged them with a low dose of empty AAV9 capsids to trigger the development of anti-AAV9 capsid antibodies. Persistent long-lived plasma cells are responsible for preexisting anti-capsid antibodies, and are sensitive to proteasome inhibition due to their high rate of immunoglobulin production.⁵⁴ Proteasome inhibition leads to the accumulation of undegraded, misfolded proteins in the endoplasmic reticulum of plasma cells, activating the unfolded protein response and eventually causing apoptosis.⁵⁵ Bortezomib, a proteasome inhibitor, is a boronic acid dipeptide derivative that reversibly binds to the 26S proteasome. When administered, bortezomib quickly distributes throughout the bloodstream and various tissues.⁵⁶ We used IMS drugs such as bortezomib to target antibody-producing plasma cells (and diminish processing and presentation of antigen), anti-CD20 antibody to deplete mature B cells, and sirolimus to inhibit antigen-induced T cell proliferation and antibody production. A combination system of bortezomib and anti-CD20 antibodies has been reported to reduce anti-AAV NAbs in mice with preexisting antibodies.⁵⁷ Bortezomib in combination with anti-CD20 antibody and sirolimus abrogated the immune response in mice by day 70 (Fig. 6).

Daratumumab, a potent antibody targeting CD38 specifically in plasma cells, has shown high efficacy in mouse models, specifically against multiple myeloma and mitochondrial transfers,⁵⁸ and is reported to reduce immunological responses to AAV vectors and allowing redosing of AAV.⁵⁹ In addition, daratumumab is an immunotherapeutic drug that activates T regulatory cells (Tregs),⁶⁰ which enhances transgene expression since endogenous regulatory Tregs are critical for tolerance induction to the transgene product and the viral vector.⁶¹ We also used daratumumab, in combination with anti-CD20 antibody and sirolimus, but this strategy led to a lower response compared with bortezomib alone or in combination with anti-CD20 and sirolimus (Supplementary Fig. S6B). This indicates that daratumumab can reduce antibodies effectively on its own and will not have a synergistic effect when combined with other IMS strategies.

Our study also demonstrated that the combination regimen of IMS enhances the efficacy of the therapeutic vector dose in wild-type mice. IMS targeting B cells, T cells, and plasma cells reduced the cellular immune response against the transgene (UFµDys1). However, further studies are required to confirm this finding because we have not performed anti-transgene antibody assays to confirm that these IMS strategies can reduce transgene antibodies.^44,62 The IMS combination treatment regimen prevented skewing of the transgene expression by preexisting capsid antibodies (Fig. 6). In cardiac tissue, we observed additional lower molecular-weight bands corresponding to microdystrophin, which were not present in skeletal muscle (Fig. 3 and 6). These signals are unlikely to arise from nonspecific detection, as they were consistently observed across replicates. Similar banding patterns have been reported with other microdystrophin constructs and reflect tissue-specific protein processing.^15,63 Global transcriptomic analysis of proteins in cardiac and skeletal muscles reveals differential expression of proteins and higher specialization of gene activity in energy production profile for contraction.⁶⁴ It has also been previously reported by our group that cardiac tissues receive more vector per cell than the skeletal muscles after AAV9 serotype transduction.⁶⁵

While the precise mechanism remains to be defined, the detection of these distinct bands raises the possibility that microdystrophin stability, maturation, or processing may differ between cardiac and skeletal muscles. Further investigation of these truncated products could provide valuable insights into optimizing construct design for cardiac and skeletal muscle correction.

Our study does have some limitations. First, we only assessed anti-AAV9 capsid antibody responses in mice. Consequently, it is possible that capsids with specific tissue tropism may generate different immune responses and may not require such potent IMS regimens to tackle adverse immune reactions. However, immune response due to the modified capsid structure could limit repeated administration and efficacy in certain patients with preexisting AAV antibodies.⁶⁶ Second, while mice are an established preclinical immunological model, the relevance of these findings to humans and other higher mammals remains to be determined. Third, our study has been designed to assess the safety and duration of combination IMS regimens in wild-type animals to tackle preexisting anti-AAV antibodies, enabling systemic therapeutic vector administration. The dose of the combination IMS regimen will likely have to be adjusted for dystrophic mice. Fourth, we did not assess the transgene-specific antibodies and whether our conditioning IMS regimen can actively suppress those antibodies. Compared with the challenge of preexisting anti-AAV capsid antibodies, transgene-related immunity is more manageable using different strategies, such as immunotolerance induction by liver-restricted transgene expression.^67,68 Fifth, the present study was conducted on the mdx mouse model maintained on a C57BL/6 genetic background, which provides proof of concept of the immunomodulatory regimen. It is important to note, however, that mouse strains vary significantly in their immune responsiveness. For example, BALB/c and DBA/2 mice are known to mount stronger humoral and cellular immune responses compared with C57BL/6. Evaluating our regimen in these strains would provide a more stringent assessment of efficacy and improve the translational relevance of the findings.

Future studies using such backgrounds will help determine whether our immunosuppressive approach can overcome immune barriers in diverse immunogenetic contexts, thereby better modeling the heterogeneity expected in patient populations. In this study, we focused primarily on suppressing humoral immune responses against the AAV capsid to enable vector administration in seropositive animals and allow for repeat dosing. While this approach effectively reduced B cell-mediated antibody responses, we acknowledge that cellular immune mechanisms, particularly T cell responses directed against the vector capsid or the transgene product, may also influence the durability and safety of gene therapy outcomes.⁶⁹ Cytotoxic T lymphocyte activation has been implicated in the decline of transgene expression and in potential hepatotoxicity in clinical trials.⁷⁰ Although assessment of T cell responses was beyond the scope of the present work, future studies will directly explore this important aspect. In particular, we plan to investigate the impact of our regimen on capsid- and transgene-specific T cell responses in follow-up studies, including upcoming work focused on hepatic-mediated immune tolerance. Understanding how both humoral and cellular immunity are modulated will be essential for optimizing the safety and efficacy of repeated AAV administration. Finally, although this approach may be promising for DMD treatment in patients with preexisting anti-AAV antibodies, potential clinical considerations would weigh the risk and benefits, including prolonged suppression of immune response, costs, and adverse effects from bortezomib and daratumumab conditioning regimens against the severity of the inherited disorder for which gene therapy is being considered.

In summary, our data indicate that UFµDys1 is a potential candidate for DMD gene therapy. Furthermore, we show that administration of a preconditioning IMS regimen consisting of anti-CD20 antibody and sirolimus allows for lower dose vector and readministration, enabling patients to receive a divided AAV dose and potentially enhance safety. We also show that a combination IMS treatment with bortezomib, rituximab (anti-CD20 antibody), and sirolimus can reduce preexisting TAbs to levels that permit systemic vector administration and therapeutic transgene production. The combined IMS treatment approach demonstrates relative safety and potential for wide-ranging gene therapy applications. However, the use of bortezomib in this protocol is not without potential adverse effects. A careful evaluation of the risk–benefit ratio is imperative, particularly when considering this approach for conditions that have alternative therapeutic options. This assessment is crucial to ensure that the potential benefits of the treatment outweigh the associated risks for each patient population under consideration.

AUTHORS’ CONTRIBUTIONS

M.S., R.B., C.A.M., K.C., P.D.T., and M.G. performed the experiments. M.S., M.C., and B.J.B. designed the experiments. M.S. interpreted the data and wrote the article. M.C., B.J.B., R.W.H., and M.B. conceptualized the study. All the authors read and approved the final article.

Footnotes

ACKNOWLEDGMENTS

The authors are grateful to Duchenne UK for their generous financial support of this project, which has played a crucial role in propelling their DMD research forward. In addition, they extend their heartfelt thanks to Claudia Mercado-Rodriguez, PhD, for her exceptional contribution to this submission. Her thorough review of both the data and article, characterized by meticulous attention to detail, has elevated the quality of this work.

FUNDING INFORMATION

No funding was received for this article.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request. GenBank accession numbers are UFµDys: PX233840 and µDys5: PX233841. The submissions are currently under review and will be made available on February 28, 2026.

AUTHOR DISCLOSURE

B.J.B. receives funding from NIH grants R01HD052682, R01AR056973, UG3NS137533, U01NS116752, and P50-AR-052646. He serves as an advisor to Rocket Pharma and Tenaya Therapeutics and is a member of both the Medical Advisory Board and the Board of Directors of the Muscular Dystrophy Association. He has received research support from Sarepta Therapeutics and Pfizer and serves on the Global Pompe Advisory Board supported by Sanofi. B.J.B. has also received consulting fees from Amicus Therapeutics, Rocket Pharma, and Tenaya Therapeutics. He is a cofounder of Ventura Life Sciences, LLC. The University of Florida is entitled to licensing revenue related to AAV technology. B.J.B. additionally serves as an uncompensated member of the Muscular Dystrophy Association Board of Directors. M.C. receives funding from NIH grants U01NS116752-01A1 and R01HD052682 and has received research support from the Friedreich’s Ataxia Research Alliance. M.C. has received consulting fees from UniQure, Voyager, Entrada, TRiNDS, and Neurogene. M.C. and B.J.B. are cofounders of Ventura Life Sciences, LLC, and the University of Florida is entitled to licensing revenue related to Pompe disease inventions. R.W.H. receives grant funding from Hoffmann-La Roche. All other authors declare no competing financial interests.

Supplemental Material

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