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. 2026 Mar 11;11:88. doi: 10.1038/s41392-026-02599-3

Phase 1 pilot study for hemophilia-A: AAV8 vector with prophylactic tacrolimus-glucocorticoid achieves therapeutic FVIII activity

Wei Liu 1,2,#, Xiaolei Pei 1,2,#, Tenghui Yu 3,#, Bingqi Xu 1,2,#, Xinyue Dai 1,2,#, Feng Xue 1,2, Lingling Chen 1,2, Xiaolin Wang 3, Yuhua Wang 1,2, Yunfei Chen 1,2, Xiaofan Liu 1,2, Rongfeng Fu 1,2, Wentian Wang 1,2, Ying Chi 1,2, Hui yuan Li 1,2, Renchi Yang 1,2, Lei Zhang 1,2,✉
PMCID: PMC12979573  PMID: 41813646

Abstract

This prospective phase 1 pilot study evaluated the safety and efficacy of GS001, an adeno-associated virus serotype 8 (AAV8)-based vector encoding B-domain–deleted factor VIII with exploratory assessment of prophylactic immunosuppression. Twelve adult severe hemophilia A participants were enrolled. Participants received GS001 at either 2×10¹² (low dose cohort) or 4×10¹² (high dose cohort) vg/kg following prophylactic treatment with prednisone alone or prednisone plus tacrolimus starting from one week before GS001 infusion. Median (range) follow-up was 156.0 (144.0-208.0) / 110.5 (104.0-130.0) weeks (2×10¹² / 4×10¹² vg/kg). Transgene expression peaked at 5-7 weeks post-infusion. In low dose cohort, at week 144.0, median (range) factor VIII activity was 5.6 (0.5-52.0) IU/dL (n = 6). In high dose cohort, at week 104.0, median (range) factor VIII activity was 42.7 (29.4-92.1) IU/dL (n = 6) and all six participants sustained factor VIII levels above 29 IU/dL. Both cohorts showed significant reductions in annualized bleeding rate and factor VIII consumption with grade 1-2 adverse events. Alanine aminotransferase elevations occurred in 3/6 and 5/6 low/high-dose participants and most were grade 1. The Single-cell RNA analysis demonstrated that the prednisone plus tacrolimus pretreatment effectively inhibited CD8+ T cell immune responses in both cohorts. Overall, GS001 infusion was safe and well-tolerated, achieving a significant increase in factor VIII level. Pre-treatment with glucocorticoids and tacrolimus may benefit by suppressing induced immune responses, thereby enhancing transgene expression without increasing the risk of infection. This study was registered at www.clinicaltrials.gov as # NCT04728841.

Subject terms: Clinical trials, Haematological diseases

Introduction

Hemophilia A (HA) is an X-linked inherited bleeding disorder caused by the deficiency or dysfunction of coagulation factor VIII (FVIII) that results in lifelong excess bleeding, typically in joints and muscles.1,2 Recurrent joint bleeding often leads to hemophilic arthropathy, characterized by chronic pain, joint deformity, and progressive disability, severely compromising patients’ quality of life. The burden of HA is substantial in China. China has a large population with an estimated 80,000 to 100,000 HA patients. The current standard care for severe HA without FVIII inhibitors is prophylactic treatment of FVIII concentrates. This approach aims to maintain a trough FVIII activity (FVIII:C) above a certain level to prevent spontaneous bleeding episodes. However, most Chinese patients receive exogenous FVIII only when they experience bleeding episodes due to economic constraints, limited access to clotting factors, or other healthcare barriers.3 Consequently, breakthrough bleeding that causes progressive joint destruction and impaired quality of life is still common.3,4 The unmet medical needs of severe HA patients in China are very significant.

Gene therapy has shown significant potential in treating hemophilia, by significantly reducing bleeding episodes, alleviating the pain associated with frequent injections, aiding in joint repair, and thus offering long-term benefits to patients. Three decades of advances in monogenic disease therapy have established adeno-associated virus (AAV) vectors as a cornerstone platform for hemophilia treatment. The liver is one of the key target organs for hemophilia gene therapy, with most serotypes of AAV primarily targeting the liver. The typical AAV vectors used in hemophilia gene therapy contain codon-optimized transgenes for F8 or F9 variants along with a liver-specific promoter. Encouraging results from several clinical trials using AAV vectors carrying F8 have been reported since 2017, including Roctavian,5–7 PF-070554808 ,and SPK8011.9 Roctavian has already been approved by European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA), and now is commercially available in certain regions.10 Extensive efforts have been made in hemophilia gene therapy to improve transduction efficiency, including higher doses of AAV (e.g., 6×10¹³ vg/kg in Roctavian), rationally designed vectors, immunosuppressants5–9,11–17 and improvement of FVIII folding, secretion, and activity.18 Great improvements have been achieved in annualized bleeding rate and proportion remaining off prophylaxis or FVIII consumption observed both in modified and wild-type AAV vectors. Despite this considerable progress, critical barriers persist, limiting the widespread and durable success of HA gene therapy. Firstly, hepatotoxicity is one of the major safety considerations. Elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level are common treatment-related adverse events (TEAEs) observed in most clinical trials, typically indicative of an immune attack on transduced hepatocytes. Secondly, host immune responses pose significant challenges. These include pre-existing immunity to AAV capsids in a substantial portion of the population, therapy-induced neutralizing antibodies (NAbs) against the AAV vector, and crucially, cytotoxic T-lymphocyte (CTL) responses that can eliminate transduced cells, thereby limiting transduction efficiency and the persistence of transgene expression.19–21 Finally, FVIII transgenes have demonstrated inferior stability compared to the longer-lasting expression often seen in hemophilia B gene therapy. This may be attributable to the larger size of the F8 transgene, the complexity of the FVIII protein, and potential cellular stress induced by its synthesis and secretion within hepatocytes.22,23

In March 2021, we initiated HA gene therapy in China using GS001 vector. As China’s first HA gene therapy trial, this study pioneers two transformative solutions: 1) GS001 (supplementary Fig. 1), a wild-type AAV8 vector with an optimized liver-specific cassette expressing B-domain-deleted FVIII24,25; and 2) prophylactic immunosuppression (prednisone alone or with tacrolimus) to alleviate immune responses triggered by AAV infusion. Our trial aimed to achieve therapeutic FVIII levels while mitigating immune challenges to HA patients through GS001’s tailored design and proactive dual immunosuppression. The vector and transgene in our study are the same as that used in GO-8 study.26 However, the promoter and polyA are totally different (supplementary materials). The GS001 promoter consists of two elements: an optimized sequence derived from the canine SERPINA1 gene and a sequence from the zebrafish ALBUMIN gene. The polyadenylation signal KS13PA originates from the common vole polyomavirus strain KS13.

The preclinical studies verified GS001’s gene transduction efficiency in F8 gene knockout mice (supplementary Fig. 2). Compared with the liver specific promoter (HLP)-Codop-FVIII-V3 vector in GO-8 study,24 GS001 demonstrated higher expression levels both in vivo and in vitro (supplementary Fig. 2a-b). In a preclinical tail vein transection (TVT) model, F8 gene knockout mice treated with GS001 exhibited reduced bleeding, comparable to mice infused with recombinant FVIII concentrate (200 IU/kg, Kovaltry, Bayer) and wild-type mice. These results indicate that the FVIII produced by GS001 has effective hemostatic activity in mice (supplementary Fig. 2d). In this clinical trial, we assessed the efficacy of GS001 in treating HA in low dose cohort (2×1012 vg/kg) and high dose cohort (4×1012 vg/kg). To further elucidate the role of tacrolimus, we compared AAV-specific T cells and AAV-specific antibody changes during gene therapy. Additionally, we validated the sensitivity of activated T cell subsets to tacrolimus post-GS001 gene therapy in vitro and analyzed the immunosuppressive effects on T cell subsets using single-cell RNA sequencing (scRNA-seq).

Results

Participant recruitment and AAV infusion dosage for HA

Between March 2021 and May 2023, 40 severe HA participants were screened. Finally, twelve participants were enrolled in two dose cohorts (six in the 2×1012 vg/kg cohort and six in the 4×1012 vg/kg cohort; Fig. 1). Participants’ demographic and baseline characteristics are presented in Table 1 and supplementary Table 1. The mean age of the participants was 27.7 ± 8.0 years (Table 1). Before enrollment, nine participants had received low dose FVIII prophylactic treatment. Prior to the screening, the mean (± standard deviation, SD) annualized bleeding rate (ABR) of these participants was 12.1 ± 5.3. Three participants had received on-demand FVIII treatment. The mean age, height, and weight of the participants were similar between two cohorts. The median (range) / mean (± SD) follow-up period was 156.0 weeks (ranging from 144.0 to 208.0) / 167.5 weeks (± 27.1) for the 2×1012 vg/kg cohort, and 110.5 weeks (ranging from 104.0 to 130.0 weeks) / 114.8 weeks (± 12.8) for the 4×1012 vg/kg cohort. All enrolled participants were included in the safety analysis set (SAS), full analysis set (FAS) and pharmacodynamic analysis set (PDS).

Fig. 1.

Fig. 1

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Trial Profile. At the time of data analysis, the median follow-up period was 156.0 weeks (range, 144.0-208.0 weeks) for 2×1012 vg/kg cohort, and 110.5 weeks (range, 104.0-130.0 weeks) for 4×1012 vg/kg cohort, respectively

Table 1.

Baseline characteristics of the participants

Characteristics Low-dose cohort (2×1012 vg/kg, n = 6) High-dose cohort (4×1012 vg/kg, n = 6)
Participant Number 1 2 3 4 5 6 7 8 9 10 11 12
Follow-up period*(weeks) 208.0 195.0 156.0 146.0 156.0 144.0 130.0 130.0 117.0 104.0 104.0 104.0
FVIII activity (IU/dL) at last follow up data 9.7 10.7 1 3.2 52.4 <1.0 33.5 98.8 34.1 43 42.3 29.4
Sex Male Male Male Male Male Male Male Male Male Male Male Male
Age (years) 21.0 19.0 18.0 37.0 25.0 31.0 24.0 23.0 35.0 35.0 42.0 22.0
Height (cm) 169 159 173 170 168 168 172 172 164 165 172 173
Weight (kg) 82 67 59 63 70 90 92 59 79 62 78.5 90
Body mass Index (Kg/m2) 28.71 26.55 19.71 21.80 24.80 31.89 31.10 19.94 29.37 22.77 26.53 30.07
Baseline FVIII:C (IU/dL) 0.3 0.2 0.2 0.5 0.3 0.3 0.4 0.3 0.2 0.1 0.3 0.2
Binding antibody against AAV8 at baseline <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10
Neutralizing Antibody against AAV8 at baseline <1:4 <1:4 <1:4 <1:4 <1:4 <1:4 1:8 <1:4 <1:4 <1:4 <1:4 <1:4
Type of FVIII replacement therapy Prophylaxis On-demand On-demand Prophylaxis Prophylaxis Prophylaxis Prophylaxis On-demand Prophylaxis Prophylaxis Prophylaxis Prophylaxis
Consumption of FVIII in previous year (IU/kg/yr) 468.0 280.0 1224.0 1144.0 4524.0 2288.0 1508.0 92.0 676.0 624.0 1612.0 286.0
Number of target joints at baseline 0 0 3 1 0 1 0 0 3 2 2 1
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*Study data cut-off date: 12 May 2025

Evaluation of gene delivery and expression efficiency (one stage clotting assay) of GS001 gene therapy for severe HA

Following GS001 infusion in the low-dose cohort, median peripheral FVIII:C rose rapidly to 50.4 IU/dL (range, 5.0-127.7 IU/dL) by one week—within the normal range (50.0-150.0 IU/dL)—and reached a peak median of 80.4 IU/dL (range, 32.9-127.7 IU/dL) at a median of 5.5 weeks (range, 1.0-7.0 weeks). (Fig. 2a-b, supplementary Fig. 3). After week 16.0, FVIII:C levels gradually stabilized at levels above 10.0 IU/dL—within the mild hemophilia range (defined as: 5.0-40.0 IU/dL)—in five of the six participants. At week 52.0, the median FVIII:C level was 7.2 (range, 1.3-54.3) IU/dL. At week 144.0, the median FVIII:C level was 5.6 (range, 0.5-52.0) IU/dL (n = 6). Among these participants, two reached mild hemophilia level (8.1 IU/dL and 10.5 IU/dL), one normal level (52.4 IU/dL), and two moderate hemophilia level (1.0 IU/dL and 3.1 IU/dL) (defined as: 1.0-5.0 IU/dL). Additionally, Participant 1 had been followed up for 208.0 weeks, and the FVIII:C was 9.7 IU/dL, which remained constant since year one (10.7 IU/dL).

Fig. 2.

Fig. 2

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FVIII:C levels (one-stage clotting assay) Post GS001 Infusion at 2×1012 vg/kg and 4×1012 vg/kg cohort. a FVIII:C levels (mean [standard deviation]). b Individual FVIII:C levels. At the time of data analysis, the median follow-up period was 156.0 weeks (range, 144.0-208.0 weeks) for 2×1012 vg/kg and 110.5 weeks (range, 104.0-130.0 weeks) for 4×1012 vg/kg cohort. FVIII:C values are with normal values of 50.0 to 150.0 IU/dL. FVIII:C levels within a 72-hour period since the last infusion of FVIII were excluded

In the 4×1012 vg/kg cohort, at one week after GS001 infusion, median peripheral FVIII:C quickly reached 146.9 (range, 131.6-198.1) IU/dL and then further increased and reached peak (288.2 (234.1-812.3) IU/dL) at 6.5 (range, 4.0-8.0) weeks (Fig. 2a-b). The median FVIII:C at week 26.0 was 105.6 (range, 74.3-132.7) IU/dL. At week 52.0, FVIII:C was 59.6 (range, 52.7-109.0) IU/dL. The median FVIII:C was non-hemophilia status (>40.0 IU/dL), which was 42.7 (range, 29.4-92.1) IU/dL at week 104.0 (n = 6). Three participants showed FVIII:C consistent with high mild hemophilia level (29.4 IU/dL, 33.5 IU/dL and 34.1 IU/dL), two participants reached a non-hemophilic state (42.3 IU/dL and 43.0 IU/dL), and one was within the normal range (98.8 IU/dL) (Fig. 2b, supplementary Fig. 3). The FVIII:C levels reported here were consistently approximately 1.3 times as high as the FVIII:C levels from the chromogenic assay (supplementary Table 2, supplementary Fig. 4).6

In the low-dose cohort, seven days before vector infusion, Participants 1-3 received prophylactic prednisone and Participants 4-6 received prophylactic prednisone treatment combined with tacrolimus. We conducted an exploratory analysis comparing FVIII:C levels between Participants 1-3 and Participants 4-6. Compared to Participants 1-3, FVIII:C of Participants 4-6 increased more rapidly and reached a higher level in seven days after vector infusion. Over the 144.0-week period, the mean FVIII:C was consistently higher in Participants 4-6 (Fig. 2b, supplementary Fig. 5-6).

Both doses of GS001 infusion significantly alleviate symptoms in HA participants

The median (range) / mean (± SD) ABR from week 3.0 to the cut-off date after GS001 infusion dropped from 11.0 (7.0-30.0) / 14.2 ± 9.3 events to 0.8 (0-10.0) / 2.6 ± 3.9 events (Fig. 3a) in the low dose cohort and from 12.5 (8.0-20.0) / 13.0 ± 4.6 to 0 (0-0.5) / 0.2 ± 0.2 events (Fig. 3b) in the high dose cohort. The median (range) / mean (± SD) annualized consumption of FVIII decreased remarkably from 1184.0 (280.0-4524.0) / 1654.7 ± 1573.4 to 177.0 (0-556.0) / 246.8 ± 259.3 IU/kg/year(y) (Fig. 3c) in the low dose cohort and from 650.0(92.0-1612.0) / 799.7 ± 628.1 to 0 (0-316.0) / (53.8 ± 128.6) IU/kg/y in the high dose cohort (Fig. 3d). None of the participants in high dose cohort experienced any bleeding events that required exogenous FVIII treatment after GS001 infusion. Two participants received low dose FVIII prophylactic treatment due to joint arthroplasty. In the high-dose cohort, one participant maintained FVIII:C of 109.0 IU/dL at 53.0 weeks post-vector infusion. Then underwent right knee arthroplasty, right hip joint arthroplasty and left knee joint arthroplasty without FVIII replacement therapy during the whole procedures. In both cohorts, all participants who received prophylaxis FVIII treatment ceased FVIII use after gene therapy.

Fig. 3.

Fig. 3

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Clinical Benefits of Participants Before and after GS001 Infusion in 2×1012 vg/kg and 4×1012 vg/kg Cohort. a, b Annualized bleeding rate. c, d Annualized dosage of FVIII infusion

Before treatment, the median (range) / mean (± SD) number of target joints in the low dose cohort and the high dose cohort was 0.5 (0-3.0) / 0.8 ± 1.2 and 1.5 (0-3.0) / 1.3 ± 1.2, respectively. This number fell to 0 (0-0) both in the low dose cohort and the high dose cohort from post-infusion to the cut-off date.

Assessment of AEs to GS001 gene therapy in severe HA

In the low dose cohort, the most common GS001 treatment related TEAEs were increased ALT and lactate dehydrogenase (LDH), both of which were reported in 3/6 (50%) participants (defined as observed value > 1× upper limit of normal) (supplementary Tables 3 and 4). All these GS001 related TEAEs were grade 1 or grade 2 in severity. One participant (Participant 6) experienced a serious AE (SAE) of increased AST (102.5 U/L, normal range: 0-50.0 U/L/) (grade 1) at week 16. At the same visit time point, his ALT was 27.7 (normal range: 0-50.0) U/L. After three days of methylprednisolone pulse therapy, AST returned to normal range. This participant experienced another SAE of increased transaminases level (ALT 296 U/L; AST 1008 U/L) (grade 4) at week 93.0, following oral treatment with benzbromarone for hyperuricaemia from week 79.0 to week 91.0. After treatment with ursodeoxycholic acid, ademetionine 1,4-butanedisulfonate, polyene phosphatidylcholine injection, and methylprednisolone, the transaminase levels returned to normal at week 97.0 (supplementary Fig. 3). The event was considered not related GS001 treatments.

In the high dose cohort, the most common GS001 related TEAE was increased FVIII:C ( > 150.0 IU/dL), reported in 6/6 (100%) participants. Most of these AEs happened in the first three weeks after GS001 treatment. Next most common were increased ALT and AST, reported in 5/6 (83.3%) and 3/6 (50.0%) participants, respectively. Participants 7 and 8 experienced SAEs of increased FVIII:C (201 IU/dL and 170.9 IU/dL) starting from days 14 and 21 after GS001 infusion (grade 1), respectively. In Participant 7, ALT increased to 3.3 times to the upper limit of normal (ULN) and was classified as a grade 2 TEAE. The increase in ALT in other participants was of grade 1 severity (Table 2 and supplementary Fig. 3). No thromboembolic events were reported. There were no deaths or GS001 related TEAEs leading to withdrawal from the study in either dose cohort.

Table 2.

TEAE summary (related to GS001, or Glucocorticoid, or Tacrolimus)

Low-dose cohort (2×1012 vg/kg) (n = 6) Participants no. (%)/Events High-dose cohort (4×1012 vg/kg) (n = 6) Participants no. (%)/Events Pooled participants (n = 12) Participants no. (%)/ Events
GS001 related TEAEs 5 (83.3) /34 6 (100) /64 11 (91.7)/98
 Grade 1 5 (83.3) /27 6 (100)/53 11 (91.7) /80
 Grade 2 1 (16.7) /7 5 (83.3)/11 6 (50.0) /18
 Grade 3 0/0 0/0 0/0
 Grade 4 0/0 0/0 0/0
 Grade 5 0/0 0/0 0/0
GS001-related SAEs 1 (16.7)/1 2 (33.3)/2 3 (25.0)/3
 Coagulation factor VIII level increased 0/0 2 (33.3)/2 2 (16.7)/2
 Aspartate aminotransferase increased 1 (16.7)/1 0/0 1 (8.3)/1
Glucocorticoid related TEAEs 5 (83.3)/38 6 (100.0)/77 11 (91.7)/115
 Grade 1 5 (83.3)/34 6 (100.0)/65 11 (91.7)/99
 Grade 2 1 (16.7)/3 6 (100.0)/12 7 (58.3)/15
 Grade 3 1 (16.7)/1 0/0 1 (8.3)/1
 Grade 4 0/0 0/0 0/0
 Grade 5 0/0 0/0 0/0
Glucocorticoid related SAE 0/0 0/0 0/0
Tacrolimus related TEAEs 2 (33.3)/4 4 (66.7)/11 6 (50.0)/15
 Grade 1 2 (33.3)/4 4 (66.7)/8 6 (50.0)/12
 Grade 2 0/0 2 (33.3)/3 2 (16.7)/3
 Grade 3 0/0 0/0 0/0
 Grade 4 0/0 0/0 0/0
 Grade 5 0/0 0/0 0/0
Tacrolimus related SAE 0/0 0/0 0/0
Tacrolimus related SAE 0/0 0/0 0/0
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Study data cut-off date: 12 May 2025

TEAE: treatment emergent adverse event. SAE: serious adverse event

Treatment related AE: AEs recorded in the eCRF as “certainly related” and “possibly related” will be considerated as AEs related to the study treatment.

A SAE is an adverse event that occurred during the study:

- Results in death.

- Life-threatening: An event where the subject faces immediate risk of death at the time of occurrence. This explicitly excludes events that would only be fatal if they had occurred in a more severe form.

- Requires unplanned inpatient hospitalization or prolongation of an existing hospitalization; This excludes planned admissions for routine treatment and visits for this study.

- Results in persistent or significant disability/incapacity;

- Results in a congenital anomaly or birth defect.

An SAE may also include any other clinically significant event that, as judged by the investigator, could compromise the patient’s well-being, or necessitate intervention to prevent the occurrence of other outcomes specified in the definition above.

All 12 participants (100%) received prophylactic glucocorticoid, and nine (75%) received prophylactic glucocorticoid plus tacrolimus. Participants 4 to 12 received tacrolimus for a median duration of 154 days (137-341 days). AEs attributed to glucocorticoids were reported by all 11 participants (91.7%). The most common events were increased white blood cell count, acne, hypokalemia, insomnia, and weight gain. AEs attributed to tacrolimus were reported by six of the 12 participants. The most common event was a mild and transient serum creatinine increase, which resolved without intervention. The following infection AEs were attributed to glucocorticoids and/or tacrolimus: skin infection, anal infection, and Epstein-Barr virus infection, each reported by one participant. All AEs except for two of anal infections were grade 1 in severity and resolved without intervention. No participants experienced SAEs attributed to glucocorticoids or tacrolimus.

Vector shedding was assessed in nine participants (Participants 4-6 and 7-12). Vector DNA was detectable in all sampled body fluids except urine and semen, peaking between day 3 and week 1 post-infusion, followed by a gradual decline. Clearance for each matrix was defined as having had negative results at three consecutive visits. Clearance occurred earliest in plasma and saliva, with slower elimination in peripheral blood mononuclear cells (PBMCs) and feces. Median time-course profiles of vector DNA levels in each dose cohort were measured (supplementary Fig. 7).

Evaluation of humoral and cellular immune responses induced by GS001 infusion in both dose cohorts

Following the establishment of the antibody and interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay detection platforms, plasma and PBMCs samples were collected from Participants 4-12. By week 1 post-infusion, all participants developed high-titer neutralizing antibodies, persisting throughout follow-up. No FVIII inhibitors were detected.

To investigate the relationship between early immune activation and the durability of transgene expression, we conducted a post hoc analysis of interferon-secreting T cell responses and anti-AAV antibody levels at Day 0 and 7 in both dose cohorts (2×10¹² and 4×10¹² vg/kg). ELISpot results (supplementary Table 5, supplementary Fig. 8a) showed no significant differences in the frequency of IFN-γ-secreting T cells between the two cohorts. However, Participant 12 in the higher-dose cohort displayed capsid-specific T cell responses, which were temporally associated with a decline in FVIII:C and elevations in ALT (supplementary Fig. 3). Plasma levels of binding and neutralizing antibodies against AAV vector also showed no significant difference between the cohorts (supplementary Fig. 8b-c, supplementary Table 6).

A higher decline rate (≥80%, from the FVIII peak to the week 52) was correlated with higher T cell counts on the day of infusion, though not statistically significant. However, by day 7, participants with a high decline rate had significantly higher specific T cell counts (supplementary Fig. 8d). No significant differences in binding or neutralizing antibody levels against AAV vector were observed between participants with high and low decline rates (supplementary Fig. 8e-f).

Therapeutic concentrations of tacrolimus inhibit the proliferation of activated CD8+ T cells from the participants infused with GS001 in vitro, but do not inhibit CD4+ T cells

To further understand tacrolimus’s immunosuppressive role during HA gene therapy, we analyzed peripheral blood PBMCs one week after therapy from Participants 8-11. PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE), activated with anti-CD3/28 beads, and treated with varying doses of tacrolimus. After 72 hours, flow cytometry was used to assess T cell viability, subset proportions, and proliferation (Fig. 4a). Post-culture, cells were stained with CD4/CD8 antibodies, and CFSE was measured to evaluate T cell subset proliferation (Fig. 4b). Different tacrolimus doses were tested for toxicity on participant PBMCs cultures, with 100 μg/mL showing significant cytotoxicity, while 10 μg/mL and 1 μg/mL showed no toxicity (Fig. 4c). In subsequent immunosuppression experiments, 1 μg/mL tacrolimus was used to study its effect on T cell proliferation. Flow cytometry results (Fig. 4d) showed that tacrolimus significantly reduced the proportion of CD8+ T cells, with minimal impact on CD4+ T cells. Analysis of CFSE levels revealed that tacrolimus significantly inhibited the proliferation of activated CD8+ T cells, without significant effect on CD4+ T cell proliferation (Fig. 4e).

Fig. 4.

Fig. 4

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Tacrolimus (TAC) specifically inhibits the activation and proliferation of T cell subsets in the peripheral blood of participants following AAV gene therapy. a Schematic workflow illustrating the detection of TAC’s immunosuppressive capability. b Flow cytometry analysis strategy, showing the representative inhibition of multiple T cell subsets proliferation after TAC treatment. c PBMCs (1×106) from hemophilia participants (Participants 8, 9, 10, and 11) post-gene therapy were treated with varying doses of TAC (low: 1 μg/mL, medium: 10 μg/mL, high: 100 μg/mL) to observe its toxic and safe dosage levels. d PBMCs (1×106) from hemophilia participants post-gene therapy were treated with an effective dose of TAC to observe changes in the proportions counts of CD4+ and CD8+ T cells. (e) PBMCs (1×106) from hemophilia participants post-gene therapy were treated with an effective dose of TAC to observe the counts of activated and proliferating CD4+ and CD8+ T cells

scRNA sequencing and analysis of PBMCs before and after GS001 infusion identify Type I interferon pathways in CD8+ T cells as potential targets for detection and intervention of specific T-cell immunity

We conducted scRNA sequencing on PBMCs from eight participants across four time points, integrating data and using Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction and clustering to identify immune cell subsets (Fig. 5a). At day 3 and 28, the percentage of T/NK cells was lower than that at day 0, while the percentage of monocytes and B cells in PBMCs at day 3 and 28 was higher than that at day 0 (Fig. 5b). Compared with day -7, the enriched scores of inflammations (Human Gene Set M5932), IFN-γ (Human Gene Set M5913) and TNFA via NFκB (Human Gene Set M5890) in CD8+ T cells from the two cohorts were found significantly suppressed by the prednisone plus tacrolimus pretreatment, with only a slight increase in IFN-α score (Human Gene Set M5911) at day 28 post-vector infusion (Fig. 5c). Differential gene expression analysis of CD8⁺ T cells (day 3 and 28 vs. day 0) identified top candidate genes (Fig. 5d; supplementary Table 7), including interferon-related genes such as DDIT4, IRF7, CD81, ISG15, PCBP1, RBM38, JUND, and STAT2. Quantitative Polymerase Chain Reaction (qPCR) validation confirmed their downregulation, which correlated with the decline of FVIII:C when samples from four participants (Participants 5-8) were stratified into rising (weeks 3-7) and declining (weeks 8–12) FVIII phases (Fig. 5e; supplementary Table 8-9). The scRNA analysis demonstrates that the prednisone plus tacrolimus pretreatment effectively inhibited CD8+ T cell immune responses in both participant cohorts. Furthermore, it identifies potential new markers for monitoring and intervention.

Fig. 5.

Fig. 5

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Analysis of Single-Cell Transcriptomics of Peripheral Blood Immune Cells Pre- and Post-Gene Therapy. a, b Single-cell transcriptome sequencing and subsequent batch integration were performed on PBMCs obtained from eight participants across four time points. The percentage of main immune cells subsets were clustered and calculated (a), both the relative frequencies and cell count of the primary single-cell clusters across all four time points were presented. The resulting data illustrate the distribution of CD8+ T cell subsets within these samples (b). c Corresponding gene enrichment scores for inflammation associated pathways are depicted at days -7, 0, 3 and 28 of infusion. The Y-axis indicates batch-corrected expression levels and enrichment scores derived from the scRNA sequencing data across multiple participants and time points. d Differential gene expression analysis was performed on CD8+ T cells from 8 participants comparing days 28 and 0 post-AAV infusion (day 28 vs day 0). e From week 1 to week 14 post-gene therapy, CD8+ cells were isolated from the peripheral blood of Participant 5, 6, 7 and 8. qPCR was utilized to determine the mRNA levels

Discussion

In this phase 1 dose-escalation study, the infusion of an F8 gene-delivered AAV8 vector, GS001, resulted in successful gene expression in severe HA patients after pre-treatment with prednisone combined with tacrolimus. Two dose levels of GS001 led to a significant increase in FVIII:C, with the magnitude of vector-derived FVIII elevation being dose-dependent. Notably, transgene was expressed as early as two days after vector administration and increased rapidly to a high level within seven days in both dose cohorts. This demonstrates an advantage in the gene delivery and expression efficiency of GS001. This early onset of expression is a promising indicator of the therapy’s potential to quickly achieve hemostatic protection. The magnitude and durability of FVIII expression are also critical metrics for evaluating gene therapy success. Mean FVIII:C (one-stage assay) at weeks 52.0 and 104.0 were 80.0 IU/dL (n = 50) and 77.9 IU/dL (n = 29, 3×1013 vg/kg) in phase 3 study of PF-07055480,27 and 63.3 IU/dL(n = 17) and 36.6 IU/dL(n = 17, 6×1013 vg/kg) in phase 3 study of Roctavian.17 In contrast, earlier-phase studies with lower doses, such as the phase 1/2 study of GO-8 (n = 3, 4×1012 vg/kg), reported FVIII:C around 20.0 IU/dL,25 and SPK-8011 (1.5 or 2×1012 vg/kg) showed a mean of 12.9 ± 6.9 IU/dL between weeks 26.0 and 52.0.9 The mean FVIII:C levels observed in our high-dose cohort (4×1012 vg/kg) at 52.0 and 104.0 weeks were 71.0 IU/dL and 48.1 IU/dL. These results suggest that GS001, even at a comparatively lower vector genome dose, can achieve robust FVIII expression that is competitive with, and in some cases superior to, other candidates in the first two years. The use of relatively low-dose vectors can also reduce production costs and enhance drug accessibility and affordability for HA gene therapy, which is particularly important in resource-constrained countries including China. Additionally, because the vector dose seems to be related to the immune response, using low-dose vectors may alleviate vector-related immune reactions. Certainly, we recognize that differences exist among products, and direct comparisons of doses should be made with caution. Given sample size is small, the efficacy is being confirmed in the phase 3 study (NCT06833983).

Regarding safety, GS001 infusion was well-tolerated in HA participants. All the ALT/AST increased in our study were less than three times the ULN, except two participants in the 4×1012 vg/kg cohort who experienced ALT/AST increased (3.3/3.1 times the ULN) respectively. Most of the ALT elevations accompanied with the decline of FVIII expression.5,17 Several clinical trials have reported that elevations in ALT coincide with declines in FVIII expression.5,17 Among the contributing factors, immune responses are considered a primary cause, including the immunogenicity of AAV vector,11,28 the infusion dose,29 and individual differences in the immune systems.30 Furthermore, the AAV vector can also trigger immune responses due to Toll-like receptor 9 (TLR9) recognizing CpG-enriched DNA sequences.31,32 Although we removed CpG motifs from our coding sequence (CDS), the inverted terminal repeats (ITRs) are CpG-enriched sequences that can lead to immunotoxicity.29,31,32

Glucocorticoid and/or other immunosuppressants treatments are common approaches to inhibit the immune response to the AAV vector. The impact of timing in administering immunosuppressants on gene therapy efficacy remains unclear and is under investigation. Our study indicates that the prophylactic use of prednisone and tacrolimus effectively reduces the number of specific T-cell responses and helps reduce the specific immune response induced by AAV infusion.33 In the low-dose group, participants receiving the combined immunosuppressive regimen exhibited faster increases in FVIII levels and higher peak FVIII levels than those receiving prednisone alone, further demonstrating tacrolimus’s unique role in modulating immune responses. The faster gene expression in participants 4-12 may also be attributed to the combined use of tacrolimus and glucocorticoids reducing T-cell immune responses against capsid protein-containing hepatocytes. However, our regimen represents a relatively novel approach in HA gene therapy, its efficacy and safety profile require further validation through additional clinical trials.

The decline in FVIII expression likely results from a combination of innate, humoral, and cellular immune factors, and our findings further support the need for integrated, longitudinal immune monitoring to clarify these dynamics. Significant elevations in ALT/AST levels indicate substantial hepatocellular damage. scRNA sequencing results suggested that IFN-α pathway-related genes in CD8+ T cells may represent novel targets for specific immune responses induced by AAV infusion. Type I interferons in CD8+ T cells are primarily implicated in influencing clonal expansion and activation through metabolic processes.34,35 Additional research indicates that the effects of Type I interferons on CD8+ T cells are STAT4-dependent.36 Our study demonstrates that elevated expression of DDIT4 and IRF7 in CD8⁺ T cells, observed across both dose cohorts, may serve as early indicators of subsequent reductions in FVIII levels. In addition, previous studies have suggested a potential role for IL-6 signaling pathways in modulating the duration of transgene expression in AAV-based gene therapy.37 These findings suggest insights for developing more targeted intervention strategies. Of course, we recognize that immune responses are not the only cause of the decrease in FVIII. There are other factors including direct transduced cell toxicity from the vector or expressed FVIII protein and gene silencing.23 In Participant 4, the reduction in FVIII levels occurred without significant elevation in transaminase levels, suggesting possible involvement of other factors. Further research, such as liver biopsy, is needed to clarify these potential contributors.

This study has several limitations. Our follow-up period is relatively short. Additionally, our immune monitoring is limited. The absence of controlled cohorts restricts direct comparative efficacy assessments. Consequently, the preliminary conclusions require further verification through studies with longer follow-up periods and more participants.

In summary, GS001 demonstrates favorable efficacy and tolerability in severe HA patients, achieving near-normal FVIII:C levels rapidly and maintaining them at clinically relevant levels through two years. By integrating a novel high-efficiency cassette and prophylactic co-administration of glucocorticoids and tacrolimus, GS001 has achieved encouraging results. This breakthrough reduces both bleeding episodes and FVIII usage in the participants. However, the durability of FVIII:C activity beyond two years post-treatment in high dose cohort remains under evaluation, as long-term follow-up data are still being collected and analyzed to fully assess the persistence ofFVIII expression.

Materials and methods

Study design and participants

This open-label, single center, dose-escalation, phase 1 pilot study evaluated the safety and efficacy of GS001 for severe HA participants. The protocol was approved by the Ethics Review Board of the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (IIT2020030-EC-2). This study was registered at ClinicalTrials.gov (NCT04728841). This trial was done in full accordance with the Declaration of Helsinki. All the participants provided written informed consent for enrollment.

Male participants (≥18 years of age) who had severe HA with FVIII:C < 1 IU/dL, no history of FVIII inhibitor development, and AAV8 neutralizing antibody titers of 1:16 or less with at least 150 days of previous exposure to FVIII concentrate were enrolled. Participants were excluded if they had known inherited or acquired thrombophilia (supplementary materials).

The Data Monitoring Committee was responsible for evaluating of safety data and gave recommendations as to whether the study could be continued or to start the escalation to next dose level.

Procedure

Eligible participants were enrolled sequentially into two dose cohorts: low dose (2×1012 vg/kg) and high dose (4×1012 vg/kg). The low-dose group received single intravenous GS001 infusion, with safety data reviewed after each set of participants before dose escalation. Following ten weeks of observation in the low-dose cohort, enrollment proceeded to the high-dose cohort.

All participants received prophylactic prednisone (1 mg/kg/day) starting one week before infusion, maintained for two weeks, and tapered from day 8 post-infusion, with a total course of 8-10 weeks. In response to persistent T cell activation despite prophylactic prednisone (supplementary Fig. 9), tacrolimus (~0.03 mg/kg/day, target trough 5–10 ng/mL) was added from Participant 4 onward and continued for approximately five months. Immunosuppression regimens were adjusted based on ALT or AST elevation (exceeding 1.5 times the baseline or the upper limit of normal, or showing a rising trend based on two consecutive tests within one week) or FVIII:C decreasing trends. Additional agents (e.g., sirolimus) could be added if necessary.

Participants were monitored (supplementary protocols) for adverse events (AE) (graded per CTCAE v5.0), FVIII:C levels, and concomitant medications. AE attribution was judged by investigator. After one year, they entered a four-year extension.

Thrombotic risk was closely monitored in participants with FVIII:C > 150 IU/dL via TAT/D-dimer levels and symptoms, with antithrombotic prophylaxis as indicated by individual risk factors.

Vector description

The vector and expressing cassette were provided by Gritgen Therapeutics. The promoter of GS001 constituted two elements: an optimized sequence derived from dog SERPINA1 gene and a sequence from zebrafish ALBUMIN gene (supplementary materials).

FVIII:C measurement

FVIII:C was assessed using both a one-stage clotting assay and a chromogenic assay. FVIII inhibitors were measured using the Bethesda assay (supplementary materials).

Outcomes

The primary objective of this study was to evaluate the safety and tolerability of different doses of GS001. Safety endpoints included TEAE, changes in ALT/AST, thrombotic events, and vector shedding of GS001 measured with qPCR (in blood, saliva, urine, PBMCs, faeces, and semen). To characterize the immune response against AAV capsid protein and FVIII transgene, the cellular immune response was assessed by monitoring FVIII:C, the ALT/AST level, and an ELISpot assay of PBMCs. A spot forming unit (SFU) more than ten per million cells and at least three folds higher than negative controls is considered positive for capsid specific T cell responses.

The secondary study endpoints included vector-derived FVIII:C, the total utilization of FVIII and the number of all bleeding events from week 3 to the cut-off date post GS001 infusion. Exploratory prespecified endpoints were changes in number of target joints from baseline.

scRNA-seq data processing

Peripheral blood (5 ml) was collected from the participants at each time point. PBMCs were freshly isolated by using a density gradient centrifugation with Ficoll-Paque and using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. PBMCs were stored at 4 °C for the subsequent generation of a scRNA library.

The 10× Genomics Chromium™ system was employed to construct the cDNA library from single cells for sequencing. This system utilizes a network of microfluidic channels to form a single-cell encapsulation system, creating gel beads in emulsion (GEMs) surrounded by oil droplets. The reverse-transcribed and amplified cDNA was constructed using 5’ gene expression and sequenced with paired-end 150 (PE150) sequencing on the NovaSeq 6000 platform (supplementary materials).

The raw sequencing data of participants were processed using Cell Ranger (version 6.1.2) with reference to the human reference genome (GRCh38) with default parameters, and data from different participants and stages were combined with Seurat (version 4.3.0) merge function. First, a total of 102 genes associated with mitochondria (13 genes), and ribosome (99 genes) were excluded and retained cells with detected gene numbers were between 200 and 7500. Subsequently, the gene counts were normalized using the NormalizeData function of Seurat for each cell. The top 30 principal components (PCs) were used to perform the data integration by invoking the RunHarmony function in Harmony2. Finally, the clustering algorithm in Seurat was utilized to cluster the cells and identifying 21 clusters (resolution=0.2).

Statistical analysis

Because of the low prevalence of HA with an estimated incidence of 1 in 5000 male births, the number of patients available for this study was correspondingly limited. The sample size was based on preliminary exploration of the safety and efficacy of phase 1 trials of gene therapy for rare diseases—eg, 3 + 3 + 3 patients in vector dose exploratory studies. Descriptive statistics were used to analyze data from all the enrolled patients, including means with SD, and medians with ranges for continuous variables and counts and percentages for categorical variables. The ABR for each patient was calculated as follows: (Number of bleeds/Total number of days during the efficacy period) × 365.25 (statistic plan).

Supplementary information

Supplementary material (1.9MB, docx)
Protocol of GS001 clinical trial (1.1MB, pdf)
Statistic plan of GS001 clinical trial (509.2KB, pdf)

Acknowledgements

We appreciated all the personnel and participants who participated in this study. This study was sponsored by Gritgen Therapeutics Co., Ltd. This study was also supported in part by the National Natural Science Foundation of China (82430010, 82470138, 82300159), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2024-I2M-ZH-016), the National Key Research and Development Program of China (2023YFC2507802), and the Tianjin Municipal Science and Technology Commission Grant (24JCZDJC33000).

Author contributions

L.Z, W.L, and T.Y, conceived and designed the clinical trial. W.L and X.P drafted the original manuscript. L.Z, and WL, participated in the study design, conducted the research, recruited participants into the trial, and analyzed and interpreted the data. X.P and B.X participated in investigating the immune response after AAV infusion, analyzing, and interpreting the data including scRNA and ELISpot assay. W.W, Y.C and H.L involved in analyzing date related to scRNA. X.W, X.D, and F.X participated in conducting the research and recruiting participants into the trial. R.Y., L.C, Y.C, and X.L and R.F were involved in recruiting participants into the trial. Y.W was involved in interpreting the coagulation related data. All authors critically revised, reviewed, and approved the final version of this manuscript.

Data availability

The study protocol is available as the supplementary appendix of this paper. In addition, data can be requested from the corresponding authors from 12 months to 36 months after study completion. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA015918) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. This work was presented in abstract form at the 66th annual meeting of the American Society of Hematology, San Diego, December 8, 2024.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Wei Liu, Xiaolei Pei, Tenghui Yu, Bingqi Xu, Xinyue Dai

Supplementary information

The online version contains supplementary material available at 10.1038/s41392-026-02599-3.

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Associated Data

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

Supplementary Materials

Supplementary material (1.9MB, docx)
Protocol of GS001 clinical trial (1.1MB, pdf)
Statistic plan of GS001 clinical trial (509.2KB, pdf)

Data Availability Statement

The study protocol is available as the supplementary appendix of this paper. In addition, data can be requested from the corresponding authors from 12 months to 36 months after study completion. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA015918) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. This work was presented in abstract form at the 66th annual meeting of the American Society of Hematology, San Diego, December 8, 2024.

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