Prevalence of Adeno-Associated Virus-9-Neutralizing Antibody in Chinese Patients with Duchenne Muscular Dystrophy

Abstract

The delivery of a mini-dystrophin gene to skeletal muscles using recombinant adeno-associated virus serotype (AAV) holds great potential as a gene therapy for Duchenne muscular dystrophy (DMD). However, the presence of anti-AAV-neutralizing antibodies (NAbs) may impede the effectiveness of gene transduction. This study aimed to evaluate the prevalence of anti-AAV9 NAbs in Chinese patients with DMD, and to characterize the target population for an AAV gene therapy. A total of one hundred male patients with DMD were included in this study, and demographic and clinical data were collected. A blood specimen was obtained from each participant for the purpose of evaluating the existence of anti-AAV9 NAbs through a cell-based functional assay conducted at a central laboratory. A NAb titer exceeding 1:4 was considered positive. The positivity rates of anti-AAV9 NAb were compared among different subgroups. The median age of this DMD cohort was 8 years old, ranging from 3 to 15 years of age. Forty-two percent of patients tested positive for anti-AAV9 NAb. Notably, all samples from patients under 4 years of age tested negative, and the positivity rates of anti-AAV9 NAb differed significantly across the three age subgroups (<4 years old, ≥4 years old and <12 years old, and ≥12 years old, χ ² = 7.221, p = 0.023). Further investigation into the living environment revealed a higher positivity rate of anti-AAV9 NAb in rural patients compared with urban patients (χ ² = 3.923, p = 0.048). Moreover, the prevalence in patients from different cities/provinces varied greatly (χ ² = 16.550, p = 0.003). There was no statistically significant difference in the positivity rate of NAb among subgroups of patients with different motor functions (ambulatory or nonambulatory) and different treatment strategies (taking or not taking glucocorticoid). In Chinese DMD patients, the prevalence of anti-AAV9 NAb was found to reach 42%. Moreover, the antibody-positive rate in children <4 years of age was low and revealed notable regional discrepancies.

INTRODUCTION

Duchenne muscular dystrophy (DMD; OMIM: 310200) is an X-linked recessive neuromuscular disease caused by mutations in the DYSTROPHIN (DMD) gene (OMIM: 300377) and occurs in ∼1 in 3,600–6,000 live male births.¹ The condition is characterized by progressive muscle weakness, leading to early death in one's 20s.¹ In recent years, glucocorticoid-centered multidisciplinary therapies have been utilized to slow down the disease's progression.² However, there is still an urgent need for therapies that target DMD gene defects.

Multiple gene therapy approaches, with the objective of restoring dystrophin, are presently being employed in clinical practice or undergoing clinical trials, such as exon skipping, stop codon readthrough, and adeno-associated virus serotype (AAV)-mediated gene-addition therapy.³ Among them, the use of recombinant AAV as a vector for delivering a mini-DYSTROPHIN gene, which encodes the smallest domain to produce functional dystrophin protein, has emerged as a promising gene therapy.^4
–6 Elevidys, a rh74 AAV-mediated gene therapy carrying truncated dystrophin, was approved by the U.S. Food and Drug Administration (FDA) this year.⁷ Nonetheless, the presence of antibodies to AAVs in the human body, induced by natural immune responses to viral infections, is likely to diminish the transduction efficiency of the recombinant AAV vectors. Several preclinical and clinical trials have shown that even relatively low-titer antibodies against AAV were associated with a lack of efficacy^8
–10 and probable safety concerns of AAV-based gene therapy.¹¹

Consequently, most gene therapies have implemented AAV seropositivity as an exclusion criterion, ascertained through transduction inhibition assays targeting neutralizing antibodies (NAbs), or alternatively, through total antibody (TAb) assays.¹² The prevalence of anti-AAV9 NAbs in the general population ranges between 4.3% and 47%,^13

–16 however, only one article has reported a positivity rate of 36% for anti-AAV9 NAbs in DMD patients¹⁷ and there is no study about the Chinese population. This study aims to evaluate the prevalence of anti-AAV9 NAb in Chinese DMD patients and various subgroups to identify the population eligible for AAV9-mediated gene therapy.

MATERIALS AND METHODS

Study population

Between November 1, 2021 and March 31, 2022, one hundred male subjects with DMD were recruited from the outpatient department of Pediatrics at Peking University First Hospital. Inclusion Criteria: Subjects were eligible to be enrolled in the study only if all the following criteria were met: (1) Chinese male subjects diagnosed with DMD based on clinical manifestations and confirmed pathogenic variation in the DMD gene. (2) Below 18 years of age. (3) Subjects and their legal guardians provided informed consent to participate in the study. Exclusion Criteria: Subjects were excluded from the study if any of the following criteria were met: (1) Current exposure to systemic immunosuppressant agents other than glucocorticoids. (2) Prior exposure to any gene therapy agent. This study protocol was approved by the Ethics Committee of Peking University First Hospital (No. 2019-233, Beijing, China).

Demographic and clinical data

Demographic data were collected, including age, ethnicity, geography (rural/urban, province of residence), and prior vaccinations. A detailed DMD medical history was also recorded, including clinical manifestation, family history of DMD, genetic testing results, current motor function, doses and regimens of glucocorticoids, and concomitant medications.

Patients were categorized based on specific criteria. Based on the living conditions of DMD, the patients were stratified into three age groups: <4 years old, ≥4 years old and <12 years old, and ≥12 years old. Specifically, children below the age of 4 predominantly reside at home, while those ages 4 and above attend kindergarten and school. After the age of 12, most patients discontinue their education due to loss of ambulatory ability. Nonambulatory status was assigned when the following definition was met: (1) full-time wheelchair use or inability to complete daily activities (such as going to the bathroom) by walking independently, based on reported performance at home by the subject or guardian, and (2) verification by a trained clinical evaluator with the inability to perform the 10-meter run/walk assessment. Ambulatory status was assigned to those able to perform the 10-meter run/walk unassisted and not requiring full-time wheelchair use or assistance with daily activities.

For subjects ≥4 years of age, the “taking glucocorticoids” treatment regimen was defined as using glucocorticoids at a stable or nonstable dose for a minimum of 3 months before study entry. “Not taking glucocorticoids” included patients who had never taken glucocorticoids or had been off glucocorticoids for at least 3 months before study entry. Given that the administration of glucocorticoids is typically initiated in children ages four or older, the definition of glucocorticoid use did not extend to children below the age of four. According to guidelines set by the National Bureau of Statistics of China, regions characterized by a resident populace surpassing 3,000 are designated as urban centers, whereas areas with a permanent population below 3,000 are categorized as rural areas, such as villages. Based on their prolonged dwelling location (>6 months), patients were categorized into urban subjects and rural subjects.

Testing of anti-AAV9 NAb

Using a cell-based approach, transduction inhibition (TI) assays detected NAbs and other nonantibody-neutralizing factors by measuring the rate of inhibition of rAAV9-mediated expression of a reporter gene with patient's serum. The details are outlined as follows. A 2 mL blood sample was obtained from each subject and placed into labeled test tubes without anticoagulants or gel separators. Serum was extracted and used for exploratory analysis of the titer of NAb against AAV9 at a central laboratory, by measuring the inhibition of the transduction of cell cultures by an AAV9 vector that carries a luciferase report gene through a luminometer. Specimens were assayed by incubating both undiluted (neat) and serial dilutions (such as 1:2, 1:4, 1:8, 1:16, …) of the serum with the AAV9 vector before the transduction of target cells. The luciferase activity was measured in the presence of the specimen and was compared with the signal generated from a control containing the AAV9 vector in the presence of the diluent alone.

The presence of anti-AAV9 NAb in the serum specimens inhibited target cell transduction and subsequent luciferase production. Qualitative results were determined based on whether the inhibition of luciferase activity at a serum dilution of 1:4 was above (positive) or below (negative) 50%. Semiquantitative results were assessed by plotting % inhibition versus log₁₀ reciprocal of the serum dilution. NAb titers were reported as the reciprocal serum dilution (1/dilution) conferring 50% inhibition (ID50) of AAV9 reporter virus transduction. Considering the wide range of NAb titers, the log₂ value is employed for comparative purposes when evaluating the variations in NAb titers across different subgroups.

Theoretically applicable gene therapy strategies

From a theoretical point of view, the gene therapy strategies are analyzed case by case. Theoretically, AAV9-mediated dystrophin-addition therapy holds potential for patients lacking anti-AAV9 NAbs or exhibiting a titer below 1:4, regardless of their specific gene mutation. Furthermore, the applicability of four commonly employed exon-skipping strategies, that is exon skipping for 51, 53, 45, and 44, was assessed. If the skipping of a particular exon can restore the reading frame disrupted by a gene mutation, the treatment approach is theoretically applicable to this mutation.¹⁸ In cases where a gene variant is amendable to multiple exon-skipping strategies, each strategy is calculated once. For example, both exon 51 and 53 skipping are applicable in the case of exon 52 deletion, and both approaches are considered in the statistical calculations. Stop-codon mutation readthrough is suitable for patients harboring nonsense mutations within the DMD gene.

Statistical analyses

Measurement data conforming to a normal distribution are expressed as mean ± standard deviation, while those not conforming to a normal distribution are expressed as median (range). Categorical data are expressed as the number of cases (%). The anti-AAV9 NAb positivity rate among different subgroups was compared using the Chi-square test or Fisher's exact probability test. The Kruskal–Wallis test was used to compare the titers of NAb in different age groups. Spearman correlation analysis was used to evaluate the correlation of NAb titer and other factors. Data processing was conducted using SPSS version 27.0 statistical software, and graphs were created using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). A two-sided p-value of <0.05 was considered statistically significant. The Bonferroni correction was utilized to account for multiple comparisons across the three age groups, resulting in an adjusted significance level of 0.05/3 = 0.017.

RESULTS

Demographic and clinical characteristics

This study involved 100 male patients diagnosed with DMD, whose demographic data, clinical characteristics, and genetic features are presented in Table 1. The patients had a median age of 8 years, ranging from 3 to 15 years. Among the patients, 32% lived in rural areas. Figure 1 illustrates a relatively broad geographical distribution of patients' residences. However, most patients lived in Beijing and its surrounding provinces, which were in proximity to our research center. Within this study, 97% of patients received timely vaccinations, with 82% of patients 4 years of age and older receiving consistent glucocorticoid therapy. In relation to the various gene mutation types, it was found that 71% of patients harbored large deletions, 11% carried large duplications, and 18% had small mutations. Notably, among the cases with small mutations, nonsense mutations constituted the majority, accounting for 61% (11 out of 18).

Figure 1.

The distribution of the residences of patients with DMD. The figure illustrates a relatively broad geographical distribution of DMD patients' residences in this study. DMD, Duchenne muscular dystrophy.

Table 1.

The demographic data, clinical characteristics, and genetic features of Chinese patients with Duchenne muscular dystrophy

Category	N = 100, n (%)
Age (years)
Median (range)	8 (3–15)
<4 years old	7 (7.0)
≥4 and <12 years old	74 (74.0)
≥12 years old	19 (19)
Geography
Rural	32 (32)
Urban	68 (68)
Motor function
Ambulatory	87 (87)
Nonambulatory	13 (13)
Treatment regimens (≥4 years), n	93
Taking glucocorticoid	76 (82)
Not taking glucocorticoid	17 (18)
Mutation type of DMD
Large deletions	71 (71)
Large duplications	11 (11)
Small mutations	18 (18)
nonsense mutations	11 (11)
Small deletions	6 (6)
Small insertions	0 (0)
Splice sites mutations	1 (1)

DMD, Duchenne muscular dystrophy.

Results of anti-AAV9 NAb testing

Out of all the samples analyzed, 42% exhibited positive results for anti-AAV9 NAb at a 1:4 sample dilution, while 49% showed positive results at a 1:1 (undiluted) sample dilution. Among samples with a titer exceeding 1.0, the titer ranged from 1.9 to 14,240, with a median titer of 284.5.

The prevalence of anti-AAV9 NAb among different cohorts

The results of anti-AAV9 NAbs were further analyzed for different age groups (Table 2). All samples were negative in patients under the age of 4, and the positivity rate of anti-AAV9 NAb exhibited statistically significant differences across the three age groups (Table 2) (χ ² = 7.221, p = 0.023). Subsequent multiple comparisons between every two groups (Fig. 2A) revealed a statistical difference in the positivity rate of anti-AAV9 NAb between patients younger than 4 years of age and those who were 12 years old or older (p = 0.010), while there was no statistical difference in the other pairings (p > 0.017). Further analysis of the living environment indicated that the positivity rate of anti-AAV9 NAb was higher in rural patients compared with urban patients (χ ² = 3.923, p = 0.048). Additionally, the prevalence of NAb in patients from different cities/provinces varied significantly (χ ² = 16.943, p = 0.007), with relatively low prevalence (<30%) observed in patients from Beijing, Tianjin, and Shanxi. There was no statistically significant difference in the prevalence of NAb among subgroups of patients with different motor functions (ambulatory or nonambulatory) and different treatment strategies (taking glucocorticoids or not taking glucocorticoids) (Table 2).

Figure 2.

The prevalence of anti-AAV9 NAb among different age groups of DMD patients. (A) Comparisons of the prevalences of anti-AAV9 NAb among DMD patients in each of the two age groups. (B) Comparisons of log₂ (titers of anti-AAV9 NAb) between the respective groups, with antibody titers below 1.0 considered as 0. The violin diagram displays the median as a solid line and the quartiles as a dashed line. Each dot represents a patient's data. In the aforementioned multiple comparisons, statistical significance was adjusted to 0.017 according to Bonferroni correction. *Comparison of <4 and ≥12-year-old age groups. AAV9, adeno-associated virus serotype 9; NAbs, neutralizing antibody; POS, positive.

Table 2.

The prevalence of anti-adeno-associated virus serotype 9-neutralizing antibody among different Duchenne muscular dystrophy cohorts

Different Cohorts	n	Positive, ^a n (%)	Negative, ^b n (%)	χ ² or Fisher's Exact Probability Test	p
Total	100	42 (42.0)	58 (58.0)
Age group (years old)				7.221	0.023
<4	7	0 (0)	7 (100.0)
≥4 and <12	74	31 (41.9)	43 (58.1)
≥12	19	11 (57.9)	8 (42.1)
Geography				3.923	0.048
Rural	32	18 (56.3)	14 (43.8)
Urban	68	24 (35.3)	44 (64.7)
Provinces/cities				16.943	0.007
Beijing	17	2 (11.8)	15 (88.2)
Hebei	13	6 (46.2)	7 (53.8)
Shandong	13	8 (61.5)	5 (38.5)
Henan	13	9 (69.2)	4 (30.7)
Shanxi	7	2 (28.6)	5 (71.4)
Tianjin	5	0 (0)	100 (5.0)
Other provinces/cities^c	32	15 (46.9)	17 (53.1)
Motor function				2.342	0.126
Ambulatory	87	34 (39.1)	53 (60.9)
Nonambulatory	13	8 (61.5)	5 (38.5)
Treatment regimens (≥4 years old)				0.133	0.715
Taking GC	76	35 (46.1)	41 (53.9)
Not-taking GC	17	7 (41.2)	10 (58.8)

Positive: The inhibition of luciferase activity at the 1:4 sample dilution was >50%.

Negative: The inhibition of luciferase activity at the 1:4 sample dilution was <50%.

Other provinces/cities encompassed all provinces or cities with fewer than five cases.

GC, glucocorticoid.

If NAb titers <1.0 were regarded as 0, the log₂ (titers of anti-AAV9 NAb) of different age groups was compared, and the difference was statistically significant (H = 6.457, p = 0.040). Further comparison between every two subgroups revealed a statistical difference in the log₂ (titers of anti-AAV9 NAb) in patients younger than 4 years of age and those who were 12 years of age or older (p = 0.014; Fig. 2B). However, considering age as a continuous variable, the correlation between age and log2 (NAb titers) does not attain statistical significance, as shown in the Supplementary Fig. S1. In patients with titers exceeding 1.0, we regarded NAb titer as a continuous variable and explored its correlation with age, geography, motor function, and treatment regimen. Regrettably, the findings yielded no statistically significant results (p > 0.05).

Theoretical applicability of various gene therapy strategies

Table 3 demonstrates the theoretical applicability of various gene therapy approaches for patients with DMD in this cohort. Notably, within this cohort, 58% of patients exhibited negative anti-AAV9 NAbs or titers below 1:4, rendering them theoretically suitable candidates for AAV-mediated gene therapy. Exon skipping therapy primarily focused on patients with large deletions, with exon 51 skipping being applicable to the highest proportion (17%) of individuals, followed by exon 45 (12%), exon 53 (11%), and exon 44 (7%). Stop-codon readthrough was applicable in the case of 11 (11%) patients harboring nonsense mutations.

Table 3.

Theoretic applicability of various gene therapy strategies to Duchenne muscular dystrophy patients in this cohort

Theoretic Applicability of Gene Therapy Strategies	All, n (%)	Large Deletions, n (%)	Large Duplications, n (%)	Small Mutations, n (%)
Theoretic Applicability of Gene Therapy Strategies	N = 100	N = 71	N = 11	N = 18
AAV9-mediated mini/micro-dystrophin delivery	58 (58)	42 (59.2)	7 (63.6)	9 (50.0)
Single exon skipping
Exon 51 skipping	17 (17)	17 (23.9)	0 (0)	0 (0)
Exon 53 skipping	11 (11)	11 (15.5)	0 (0)	0 (0)
Exon 45 skipping	12 (12)	11 (15.5)	1 (9.1)	0 (0)
Exon 44 skipping	7 (7)	7 (9.9)	0 (0)	0 (0)
Stop-codon readthrough	11 (11)	0 (0)	0 (0)	11 (61.1)

AAV9, adeno-associated virus serotype 9.

DISCUSSION

AAV-mediated gene therapy has been employed in the treatment of inherited neuromuscular disorders.^19,20 For instance, Onasemnogene abeparvovec (Zolgensma), a gene therapy that employs AAV9 as a vector, has been approved for treating patients with spinal muscular atrophy.²¹ In addition, Elevidys, a rh74 AAV-mediated gene therapy carrying truncated dystrophin, was approved by the FDA this year as the first rAAV-based therapy for patients with DMD.⁷ Several clinical trials (NCT Nos. 03368742, 04240314, and 03362502) are also underway to explore the efficacy and safety of AAV-mediated gene therapy in patients with DMD.^4
–6 Previous studies have demonstrated that preexisting anti-AAV antibodies can impede the effectiveness of AAV-based gene therapy.^8
–10 Furthermore, high antibody titers may trigger a stronger host immune response, potentially leading to severe adverse reactions.¹¹ Therefore, screening for antibodies against AAV is crucial before considering AAV-based therapies. To date, only one article has been published to report the prevalence of anti-AAV9 NAb in U.S. patients with DMD.¹⁷

This study aims to investigate the seroprevalence of anti-AAV9 NAb in Chinese DMD patients, and to characterize the target population for an AAV gene therapy.

Among AAV antibodies, NAbs can bind to the AAV capsid and inhibit vector transduction, while non-NAbs bind to the capsid but do not impede vector transduction. AAV antibody detection methods are diverse and have not been standardized.^22,23 TI assays employ cell-based approach, to detect anti-AAV NAbs and other nonantibody-neutralizing factors by measuring the extent of AAV TI by patient serum, while TAb assays detect both NAbs and non-NAbs.¹² Both TI assays and TAb assays enable semiquantitative results through series dilutions of serum or obtain qualitative results according to a set threshold. For instance, if the threshold is set at 1:4, positive indicates >50% inhibitory transduction at the dilution of 1:4. TI assays can directly reflect the inhibition of transduction, however, the experiment procedure is complicated. Conversely, TAb assays is comparatively simple.¹²

Currently, the correlation between the outcomes obtained through TI assays and TAb assays remains ambiguous.¹² Additionally, if both antibody tests are required to be negative in enrollment, it would narrow the population who can benefit from gene therapy. Therefore, in the future, it may be necessary to standardize anti-AAV antibody detection methods and establishing relatively consistent positivity thresholds.

We reviewed the positive rates of AAV antibodies reported in previous literature and summarized them in Table 4.^{14

–17,24

–30} In this study, the prevalence of anti-AAV9 NAbs in DMD patients was 42%, which is slightly higher than the prevalence reported by Verma et al.¹⁷ (36%) in DMD patients in Atlanta, United States, and the prevalence reported by Mimuro et al.¹⁵ in normal controls (36.5%) and hemophilia patients (27.4%) in Japan. However, the prevalence of NAbs varies significantly across different regions of China, with the prevalence among patients in Beijing and Tianjin being <30%, while the prevalence among patients in Shandong and Henan being >60%. Moreover, the prevalence of NAbs among rural patients was higher than that among urban patients. This regional variation in the prevalence of anti-AAV NAbs is consistent with findings in the literature²⁹ and is thought to be related to the different prevalences of AAV in different regions.

Table 4.

Summary of seroprevalence of anti-adeno-associated virus serotype antibodies reported in previous literature

Study	Type of Abs	AAV Serotype	Individuals (n)	Seroprevalence	Influencing Factor(s)
Verma et al.¹⁷	NAbs	AAV2, 8, 9, and rh74	Male patients with DMD (101)	AAV2 56%, AAV8 47%, AAV9 36%, AAVrh74 32%	1. Age was not correlated with NAb titer.2. Ambulatory participants had higher seroprevalence.3. Different prevalence among different ethnic groups
Sierra-Delgado et al.²⁴	NAbs	AAV1, 2, 9	Patients with HF (60);Healthy individuals (60)	HF: AAV2 43%, AAV1 33.3%, AAV9 20%Control: AAV2 45%, AAV1 33.3%, AAV9 23.2%	1. HF group exhibited more common combined seropositivity.2. Exposure to toxins was associated with the presence of NAb
Aharoni et al.²⁵	TAbs	AAV9	Children with 5q SMA (<2 years old) (34)	AAV9 17.6%
Stolte et al.¹⁶	TAbs	AAV9	Adult patients with SMA types 2 and 3 (69)	AAV9 4.3%	Seroprevalence was not correlated with age, sex, SMA type, ambulatory condition, or ventilatory status
Klamroth et al.²⁹	TAbs	AAV2, 5, 6, 8, and rh10	Participants with hemophilia A (546)	AAV2 58.5%, AAV5 34.8%, AAV6 48.7%, AAV8 45.6%, AAVrh10 46.0%	For all serotypes, seropositivity tended to increase with age
Day et al.²⁶	TAbs	AAV9	Patients with SMA type 1 (196);Biological mothers (155)	SMA: AAV9 7.7%Biological mothers: AAV9 14.8%
Kruzik et al.²⁷	NAbs	AAV1, 2, 5, 8	Patients with hemophilia B (29)Healthy donors (120)	Hemophilia B: AAV8 41%Healthy: AAV1 27%, AAV2 47–74%, AAV5 20–59%, AAV8 32–63%	There were regional differences in seroprevalence
Fu et al.¹⁴	TAbs	AAV1–9 and rh74	MPS III patients (38)Healthy controls (35)	MPS IIIA: AAV1–9 13–33%MPS IIIB: AAV1–9 7–14%Healthy: AAV1–9 11–37%	Seroprevalence for the majority of tested AAV serotypes appears to peak before 8 years of age in MPS III subjects
Ferla et al.²⁸	NAbs	AAV8	MPS VI patients (36)	AAV8 42%
Mimuro et al.¹⁵	NAbs	AAV1, 2, 5, 8, and 9	Hemophilia patients (59)Healthy subjects (85)	Healthy: AAV1 36.5%, AAV2 35.3%, AAV5 37.6%, AAV8 32.9%, AAV9 36.5%Hemophilia patients: AAV1 39.7%, AAV2 28.8%, AAV5 35.6%, AAV8 32.9%, AAV9 27.4%	High titers of NAbs were more evident in older individuals (≥42 years old)
Roberto Calcedo et al.³⁰ (2011)	NAbs	AAV2, AAV8	Anonymous human subjects of different age groups (752)	At birth: AAV2 59%, AAV8 36%;Infants (ages <1 year): 15%; Toddlers (ages 1 to 3 years): 14%;Older children (ages 3–18 years): 21%	Prevalence of NAbs to both AAV serotypes declined significantly after birth, reaching a nadir in the 7- to 11-month group, and then progressively increases through childhood and adolescence

AAV, adeno-associated virus; Abs, antibodies; HF, heart failure; MPS, Mucopolysaccharidosis; NAbs, neutralizing antibodies; SMA, spinal muscular atrophy; TAbs, total antibodies.

Beyond geographic variables, the interaction between age and AAV antibody positivity rates was also noticed, although prior investigations have yielded equivocal findings. Verma et al.¹⁷ tested anti-AAV9 NAbs in 101 patients with DMD and found no correlation between age and NAb titer. However, in our study, we found that both NAb positivity rate and log₂ (titer) differed across different age groups, which is consistent with Klamroth et al.'s²⁹ recent study on the seroprevalence of AAV antibodies in people with hemophilia A. Notably, all children under 4 years of age in our study tested negative for NAbs, and previous studies also found that the antibody positive rate was low between 6 months and 3 years of age, suggesting that younger patients may be more suitable for AAV-mediated gene therapy. We observed no statistical difference in anti-AAV9 NAb positivity rate between different subgroups of glucocorticoid use conditions, which was consistent with previous literatures. However, due to the limited sample size, future studies with larger samples are needed to explore risk factors for NAb positivity.

Moreover, this study explored the theoretical feasibility of diverse gene therapy approaches within this cohort. Among the exon skipping therapies, exon 51 skipping exhibited the highest theoretical applicability of 17% among DMD patients, aligning with the previously reported proportion of 13–20%.^18,31,32 Conversely, skipping of exon 53, 45, and 44 demonstrated applicability to only ∼10% of patients each. The stop-codon readthrough approach exclusively applied to patients with nonsense mutations, which account for about 10% of DMD gene mutations.^31,32 Despite 42% of patients in this study exhibiting anti-AAV9 NAbs titers more than 1:4, 58% of the patients remained eligible candidates for AAV-mediated gene therapy. This highlights the potential of AAV-mediated gene therapy to benefit a larger population of DMD patients, particularly younger patients, given the lower prevalence of anti-AAV9 Abs.

Additionally, innovative approaches are currently under development to tackle pre-existing immune reactions against AAV, with the aim of expanding the benefits to a wider patient population.²⁰ One approach involves mitigating immune clearance of the vector or minimizing susceptibility to NAb. This can be achieved through tactics such as targeted organ injection, administering high doses of AAV, employing an empty vector to sequester NAbs, and modifying the AAV structure to decrease susceptibility to NAb. Another type of strategy is to suppress the immune response before drug administration, such as using plasma exchange, immunosuppressive agents, immunoglobulin G (IgG)-degrading enzymes (immunoglobulin G-degrading enzyme of Streptococcus pyogenes or immunoglobulin G-degrading enzyme of Streptococcus equi, a cysteine protease known for its ability to selectively lyse IgG) to reduce the titer of NAb.

Limitation

This study employed a continuous enrollment approach without conducting random sampling in each region, and the limited sample may impede the representativeness of anti-AAV9 NAb levels across each region. Moreover, the NAbs were tested only once in all patients, so there was no exploration of the dynamic changes in NAb levels in this study. Hence, it is necessary to conduct more extensive studies with larger samples and longer duration to address these limitations.

CONCLUSION

This study demonstrated a seroprevalence of 42% for anti-AAV9 NAbs in Chinese individuals with DMD. The lower prevalence of NAbs observed in younger patients renders them promising candidates for AAV-mediated gene therapy. Additionally, notable regional disparities were observed in the prevalence of anti-AAV9 NAbs, while no statistically significant variations were identified in the positivity rate of NAbs among different subgroups based on glucocorticoid use conditions and walking ability.

Footnotes

ACKNOWLEDGMENTS

Pfizer Investment CO. LTD. sponsored this study with the aim of comprehending AAV9 NAbs in Chinese DMD patients to identify targeted population suitable for AAV gene therapy. However, the design, recruitment, data analysis, and writing of the study were carried out independently by the investigators without interference from Pfizer. The authors are grateful to our research participants and their families. ChatGPT version 3.5 was employed for linguistic refinement; however, the polished outcomes underwent manual verification to ensure precise expression of the author's intended meaning.

AUTHORs' CONTRIBUTIONS

C.J.W., D.L.L., M.Z., Y.P.Z., Y.D.L., Y.B.F., L.W., and J.Y.L. collected the data. C.J.W. wrote the article. H.X., Y.W.J., and X.Z.C. designed the study and revised the article. All authors contributed to the article and approved the submitted version.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author.

ETHICS STATEMENT

This study was approved by the Ethics Committee of Peking University First Hospital (No. 2019-233). The participants provided written informed consent before their involvement in the study and signed agreements for the publication of this article.

AUTHOR DISCLOSURE

The author declares that the research was conducted in the absence of other commercial or financial relationships that could be construed as a potential conflict of interest.

FUNDING INFORMATION

This study was supported by funding from Pfizer Investment CO. LTD. and grants from the National High-Level Hospital Clinical Research Funding (High-Quality Clinical Research Project of Peking University First Hospital, No. 2022CR69; Interdepartmental Research Project of Peking University First Hospital, No. 2023IR51), the Natural Science Foundation of Beijing Municipality (No. 7212116), the National Natural Science Foundation of China (No. 82171393), National Key Research and Development Program of China (No. 2016YFC0901505), Peking University First Hospital Research Seed Fund (2019SF06), and Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases (No. BZ0317).

Supplementary Material

Supplementary Figure S1

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