Abstract
Purpose
No effective treatment for leber hereditary optic neuropathy (LHON) caused by ND1 mutation is available.This study evaluated the safety and efficacy of a single unilateral intravitreal injection rAAV2-ND1 in various doses for the treatment of LHON.
Methods
Twelve patients with LHON (ND1 mutation) in two groups with six participants each.The low-dose group received injection of rAAV2-ND1 in a dose of 1.5 × 108 vg/eye while the high-dose group received 1.5 × 109 vg/eye.The safety endpoint was the incidence of adverse events (AEs).The primary efficacy endpoint was changes of best corrected visual acuity (BCVA).The secondary efficacy endpoints were improvement in visual field (VF), visual field index (VFI), and mean deviation (MD) and change in retinal nerve fiber layer (RNFL) thickness.
Results
In total,11 mild eye-related AEs occurred in the participants in both groups, and short-term drug treatment returned to normal.The difference was statistically significant in BCVA of the injected eyes in the low-dose group between 12 months after treatment and at baseline.The differences in BCVA of the uninjected eyes in the high-dose group between baseline and 3 months or 6 months after treatment were statistically significant.At 12 months after treatment, the rate of improvement in BCVA for the injected eyes in the low-dose groups was 66.7% (4/6),while BCVA for the uninjected eyes in the high-dose groups was 50.0% (3/6),and patients in both groups had binocular VF (VFI, MD) and RNFL thicknesses that did not significantly differ from baseline.
Conclusion
Preliminary clinical evidence shows that rAAV2-ND1 ophthalmic injection is a safe and effective treatment for LHON due to ND1 mutation.
Trial Registration
Trial registration number: ChiCTR2000041574, Date:12/29/2020.
Keywords: RAAV2-ND1, Gene therapy, Leber hereditary optic neuropathy, Mitochondrial disease
Key messages
What is known
Currently, the focus on Leber hereditary optic neuropathy (LHON) caused by the most common ND4 gene mutation, whereas little is done on LHON caused by the less common ND1 mutation.
What is new
Gene therapy LHON ((ND1 mutation)) is safe and effective.
Monocular gene therapy, LHON patients can improve binocular vision function.
Gene therapy needs to further explore the optimal therapeutic titer, not the higher the titer, the better the therapeutic effect.
Introduction
Leber hereditary optic neuropathy (LHON) is a common maternally inherited mitochondrial disease caused by mutations in mitochondrial DNA (mtDNA).LHON was first described in 1871 by Theodore Leber, a German ophthalmologist [1]. It usually affects young men.The clinical manifestation of LHON is pain-less acute or sub-acute central vision loss, either simultaneous or successive in both eyes [2, 3]. The three primary pathogenic variants associated with LHON are m.11778G > A,m.3460G > A,and m.14484T > C,which are associated with reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 (ND4),subunit 1 (ND1),and subunit 6 (ND6) of complex I in the electron transport chain, respectively.The m.3460G > A variant accounts for approximately 13% of all cases of LHON [4]. Mutations in these genes alter the function of mitochondrial respiratory chain proteins, resulting in decreased adenosine triphosphate (ATP) synthesis and increased free radical, reactive oxygen species production, and oxidative stress in optic nerve cells, which lead to neuronal damage and retinal ganglion cell death and ultimately, a reduced or even complete loss of vision [5, 6]. Currently, no effective treatment for LHON is available.
Currently, the focus is on LHON caused by the most common ND4 gene mutation, whereas little is done on LHON caused by the less common ND1 mutation.In 2011,we initiated a clinical trial on rAAV2-ND4 gene therapy for LHON caused by the m.11778G > A mutation [7–13]. Now, we aim to study LHON caused by the ND1 mutation using our previous finding as a foundation.
After a series of preclinical studies and preliminary confirmation for drug safety and efficacy, in 2021,we conducted a clinical trial including a single unilateral intravitreal injection of recombinant adeno-associated virus type 2 human ND1 (rAAV2-ND1) in 12 patients with a 12-month follow-up period.We report the results of our trial here.
Methods
Construction of rAAV2-ND1 vector
The ND1 sequence in rAAV2-ND1 vector has been codon optimized, which can improve the expression level of ND1 mRNA and protein after codon optimization.It was constructed by Neurophth Biotechnology Co.,Ltd.,Wuhan, China.The promoter of rAAV2-ND1 vector is CMV, and the DNA coding sequence contains COX10 MTS-ND1-Cox10 3 ‘UTR, which is divided into two parts, COX10 MTS-ND1 (sequence 1) and COX10 3’ UTR (sequence 2).The COX10 MTS-ND1 gene sequence was connected to the kanamycin skeleton vector by the method of enzyme ligation.The COX10 MTS sequence length was 84 bp, the ND1 sequence length was 957 bp, and the 3 ‘UTR sequence length was 1425 bp.COX10 MTS gene is a mitochondrial targeting sequence, guiding ND1 protein into the mitochondria to exert its physiological function.COX10 3 ‘UTR is a non-coding region, designed behind ND1 protein, and its role is to stabilize the expression of mitochondrial targeting sequence and ND1.
Patients
The inclusion criteria of this study were shown as follows: (1)Mutation in the LHON-ND1 locus diagnosed by genetic testing (3460G > A,3700G > A,3635G > A,3733G > A,4136 A > G,3571 C > T,etc.);(2)Because LHON has a 22% rate of spontaneous visual recovery [14], the duration of observation of the natural course of disease must be over one year and the patient must not have undergone any other treatment in the last three months, and best corrected visual acuity (BCVA) must not have improved, to ensure that any improvement in BCVA after treatment is the effect of gene therapy alone; (3)Binocular fractional visual acuity < 0.3 (> 0.5 logMAR); (4) age 12–65 years, either gender; (5)able to tolerate local anesthetic surgery; (6)agreed not to use other drugs or treatments that may target the disease during the treatment period; and (7)voluntary participation and patient and/or his or her legal representative gave informed consent in writing.
The exclusion criteria of this study were shown as follows: (1)concomitant chronic diseases, such as diabetes mellitus, severe cardiovascular, cerebrovascular, hepatic, renal, orprimary disease; (2)other systemic neurological diseases, recovering from heart surgery, with psychiatric disorders or cancer; (3)other eye diseases affecting vision, such as glaucoma; (4)participation in other clinical trials within the last three months; (5)a history of alcohol, tobacco, drugs, or substance abuse, or exposure to toxic substances; (6)pregnant or lactating women, or those planning to become pregnant within 12 months; and(7)significantly abnormal results in preoperative immunological tests (e.g.,CD3/CD8).
Trial design
In this study, a prospective, open-label, single-center, controlled design was used to select the eye with poorer BCVA as the injected eye (IE) for single intravitreal injection (when bilateral BCVA was equal, the right eye was selected as the IE); the contralateral eye was defined as the uninjected eye (UE).Using a controlled assignment,12 patients were divided into two groups with six patients each.The vitreous humor of the IE was injected with 0.05 mL rAAV2-ND1 ophthalmic solution, with a low dose of 1.5 × 108 vg/eye in the first group and a high dose of 1.5 × 109 vg/eye in the second group.Then, we observed and compared the safety and efficacy of two different doses of this drug.The doses were chosen with reference to prior clinical trials of ND4 mutation-associated LHON [7] and our animal studies on ND1 mutation-associated LHON.
The primary endpoint of the study was the changes of BCVA after 12 months treatment.The secondary endpoints were improvement in visual field (VF), visual field index (VFI), and mean deviation (MD) and changes in retinal nerve fiber layer (RNFL) thickness after 12 months treatment.
Registration, informed consent, and ethical approval
The trial protocol was approved by the Institutional Review Board of Shiyan Taihe Hospital, Hubei University of Medicine (approval no.: 201919) and registered with the Chinese Clinical Trials Registry (registration number: ChiCTR2000041574;registration date: December 2020).All participants and their guardians provided informed consent in writing.The study was conducted in accordance with the Declaration of Helsinki with respect to informing participants of the purpose and procedures of the trial, as well as its associated risks.
Safety and efficacy
General examination: All patients underwent a detailed systemic examination before treatment, including complete blood count, urinalysis, liver and kidney function tests, coagulation tests, infectious disease screening, immune function tests (humoral and cellular immunity), electrocardiogram, and chest fluoroscopy.Cellular immunity is the collection of patients’ venous blood and human T-lymphocyte subsets, namely CD3+ (normal range,50–84%),CD3+/CD4+ (normal range,27–51%) and CD3+/CD8+ (normal range,15–44%),were counted.Humoral immunity is the detection of complement C3 and C4 in human venous blood.
Eye examination: Basic ocular examinations included visual acuity, intraocular pressure (IOP), and slit-lamp microscopic examination of the anterior segment of the eye.Special examinations included fundus photography, optical coherence tomography (OCT), and visual field examination, which were performed by the same ophthalmologist before gene therapy and at 1, 3, 6 and 12 months after treatment.
BCVA was examined using an international standard 4 m Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart (Precision Vision, USA) with one row of 0.1 logMAR units (5 letters), with higher values indicating poorer vision.A change in BCVA equal to or greater than 0.3 logMAR was considered a significant improvement.The examination was repeated three times every 10 min to confirm the improvement in visual acuity, and the average was used as the final BCVA.
A Humphrey field analyser (Carl Zeiss 740i, Carl Zeiss, Dublin, CA, USA) was used for the visual field test.The testing procedure included the 30 − 2 central threshold test and SITA fast.The major recorded parameters were VFI and MD.A 10% increase in VFI or a 3dB decrease in MD was defined as improvement.
Spectrlis HRA + OCT(Heidelberg Engineering, Heidelberg, Germany) Analysis of RNFL.The thickness of RNFL in the upper, lower, nasal and temporal quadrants, as well as the retinal thickness in the macular area, were scanned in a range of 6 × 6 mm.
Perioperative hormonal medication: To prevent possible serious adverse events during and after treatment, the 12 patients were placed on a 3-week course of 30 mg/60 kg/day oral prednisone for 7 days before surgery, 60 mg/60 kg/day on the day of surgery and for 2 days after surgery,50 mg/60 kg/day for days 4–6 after surgery, 40 mg/60 kg/day for days 7–9 after surgery, and 30 mg/ 60 kg/day for days 10–14 after surgery.
Statistics
IBM SPSS 22.0 software was used for statistical analysis of data.An α value of 0.05 was selected as the level of significance.Count data were expressed as frequencies and percentages, and measurement data were expressed as mean ± standard deviation (mean ± SD).Paired t-test was used to compare the BCVA, VF (VFI, MD), and RNFL thickness in the injected and uninjected eyes in the two groups at baseline and at 1,3,6 and 12 months after treatment; repeated measures ANOVA was used for inter-group comparison.Differences with p < 0.05 were considered statistically significant.
Results
Safety evaluation and adverse events
Over the 12-month follow-up period, six adverse events(AEs) occurred in the low-dose group and five AEs occurred in the high-dose group.A total of 11 mild eye-related AEs occurred in the two groups, all in the injected eye.These included nine cases of postoperative conjunctival hyperemia, which resolved spontaneously after 1 week, and two cases of mildly elevated IOP (24 mmHg and 25 mmHg) after operation, both of which returned to normal after the discontinuation of topical tobramycin-dexamethasone.There were no serious complications, such as intraocular inflammation, cataract, retinal detachment, or vision loss.No damage to the retina (Fig. 1) or other ocular tissue was observed, and no other abnormalities were observed on systemic examination.
Fig. 1.
Fundus photography obtained before and after the intravitreal injection of rAAV2-ND1 (Low-dose group: Patient No.1–6; High-dose group: Patient No. 7–12). A representative 30° fundus photograph showing the optic disc and macula of injected eyes before and at 12 months after intravitreal injection. The retinal structure appears normal in all photographs with no apparent abnormalities
General characteristics of patients
Twelve participants (10 with the 3460G > A mutation and 2 with the 3700G > A mutation), all male, were included in the study since January 2021.The ages of the patients in the low- and high-dose groups at the time of gene therapy were 25.33 ± 7.50 years and 23.00 ± 3.10 years, respectively, and the duration of disease was 55.67 ± 17.60 months and 37.50 ± 22.97 months, respectively.Two sample t-test showed that the differences in age, duration of disease, and BCVA of patients in the two groups before treatment were not statistically significant (p > 0.05) and were comparable to one another.The general characteristics of the patients in the two groups are shown in Table 1.
Table 1.
General characteristics and BCVA (logMAR) of the two groups of patients before treatment and 12 months after treatment
| Patient | Gender | Eye | Age(year) | BCVA before treatment | BCVA after treatment | |||
|---|---|---|---|---|---|---|---|---|
| Onset | Gene therapy | IE | UE | IE | UE | |||
| Low dose group (1.5 × 108 vg/eye) | ||||||||
| 1 | Male | Right | 25 | 30 | 1.4 | 1.2 | 1.1(−0.3) | 1.1(−0.1) |
| 2 | Male | Right | 17 | 23 | 2 | 2 | 1.7(−0.3) | 2(0) |
| 3 | Male | Right | 20 | 22 | 1.5 | 1.5 | 1.2(−0.3) | 1.3(−0.2) |
| 4 | Male | Right | 27 | 31 | 2.3 | 2 | 2(−0.3) | 2(0) |
| 5 | Male | Left | 8 | 13 | 1 | 0.8 | 1(0) | 0.7(−0.1) |
| 6 | Male | Right | 29 | 33 | 1.3 | 1.2 | 1.3(0) | 1.3(+ 0.1) |
| Average | 21 | 25.3 | 1.58 | 1.45 | 1.37(−0.21) | 1.4(−0.05) | ||
| High dose group (1.5 × 10 9 vg/eye) | ||||||||
| 7 | Male | Right | 25 | 26 | 2 | 2 | 1.7(−0.3) | 1.7(−0.3) |
| 8 | Male | Right | 20 | 26 | 2.3 | 2.3 | 2.3(0) | 2.3(0) |
| 9 | Male | Right | 17 | 20 | 2 | 2 | 2(0) | 2(0) |
| 10 | Male | Right | 16 | 19 | 0.8 | 0.7 | 0.5(−0.3) | 0.3(−0.4) |
| 11 | Male | Right | 21 | 22 | 0.8 | 0.6 | 0.7(−0.1) | 0.1(−0.5) |
| 12 | Male | Left | 22 | 25 | 1.7 | 1.5 | 1.5(−0.2) | 1.4(−0.1) |
| Average | 20.2 | 23 | 1.6 | 1.52 | 1.45(−0.15) | 1.3(−0.22) | ||
Best corrected visual acuity (BCVA)
Paired t-tests were performed to compare BCVA at baseline and at 1,3,6 and 12 months after treatment (Table 2).The difference in BCVA of IE in the low-dose group between 12 months after treatment and at baseline (1.37 ± 0.40 logMAR vs. 1.58 ± 0.48 logMAR) was statistically significant (p = 0.042).The differences in BCVA of UE in the high-dose group between 3 months after treatment and at baseline (1.28 ± 0.83 logMAR vs. 1.52 ± 0.72 logMAR) and between 6 months after treatment and at baseline (1.23 ± 0.78 logMAR vs. 1.52 ± 0.72 logMAR) were both statistically significant (p < 0.05);no other differences were statistically significant.There was no significant difference in BCVA in IE and UE between the two groups at baseline and at 1,3,6 and 12 months after treatment (Fig. 2A, B).
Table 2.
The difference in BCVA in IE and UE between at baseline and at 1, 3, 6 and 12 months after treatment
| Dose | Eye | Baseline | After treatment | |||
|---|---|---|---|---|---|---|
| 1 month | 3 months | 6 months | 12 months | |||
| Low-dose | IE | 1.58 ± 0.48 | 1.50 ± 0.55(0.669) | 1.50 ± 0.41(0.658) | 1.45 ± 0.44(0.324) | 1.37 ± 0.40(0.042*) |
| UE | 1.45 ± 0.48 | 1.43 ± 0.55(0.999) | 1.38 ± 0.45(0.810) | 1.45 ± 0.47(1.000) | 1.40 ± 0.51(0.945) | |
| High-dose | IE | 1.60 ± 0.65 | 1.50 ± 0.64(0.517) | 1.43 ± 0.63(0.116) | 1.48 ± 0.56(0.441) | 1.45 ± 0.71(0.205) |
| UE | 1.52 ± 0.72 | 1.32 ± 0.88(0.094) | 1.28 ± 0.83(0.023*) | 1.23 ± 0.78(0.001*) | 1.30 ± 0.91(0.062) | |
(*): Differences with p < 0.05 were considered statistically significant
Fig. 2.
At 1, 3, 6, and 12 months after intravitreal injection of rAAV2-ND1, the mean BCVA of injected eyes (A) and uninjected eyes (B) in the two groups improved from baseline. Before and after intravitreal injection of rAAV2-ND1, the mean VFI 、MD and RNFL thickness changes of injected eyes (C, E, G) and uninjected eyes (D, F, H) of the two groups of patients
Twelve months after treatment, the rates of improvement in mean BCVA for IE in the low- and high-dose groups were 66.7% (4/6) and 33.3% (2/6) respectively, at improvements of 0.22 logMAR and 0.15 logMAR from baseline, respectively.The rates of improvement in mean BCVA for UE in the low- and high-dose groups were 0.0% (0/6) and 50.0% (3/6),respectively, at improvements of 0.05 logMAR and 0.22 logMAR from baseline, respectively (Tables 2 and 3).
Table 3.
The rates of improvement in mean BCVA for IE and UE in the low- and high-dose groups
| Evaluation criteria | Low dose group | High dose group | ||
|---|---|---|---|---|
| IE | UE | IE | UE | |
| Gain of ≥ 3 ETDRS lines | 4(66.7%) | 0(0.0%) | 2(33.3%) | 3(50.0%) |
| Within 3 lines | 2(33.3%) | 6(100.0%) | 4(66.7%) | 3(50.0%) |
| Loss of ≥ 3 lines | 0(0.0%) | 0(0.0%) | 0(0.0%) | 0(0.0%) |
Injected eye
Uninjected eye
Visual field
Different doses of gene therapy had no statistically significant effect on the mean VFI of IE and UE between the two groups (Fig. 2C, D).In the low-dose group, the change in mean VFI from before treatment to 12 months after treatment was from 25.67 ± 18.08% to 26.33 ± 22.37% in IE and from 35.17 ± 30.29% to 30.17 ± 29.82% in UE.In the high-dose group, the change in mean VFI from before treatment to 12 months after treatment was from 30.33 ± 40.05% to 38.83 ± 38.53% in IE and from 36.50 ± 39.82% to 38.50 ± 39.16% in UE.No changes were statistically significant (p > 0.05).
Different doses of gene therapy had no statistically significant effect on the mean MD of IE and UE between the two groups (Fig. 2E, F).In the low-dose group, the change in mean MD from before treatment to 12 months after treatment was from − 22.7 ± 5.22 dB to −23.13 ± 6.78 dB in IE and from − 21.39 ± 7.46 dB to −22.51 ± 8.64 dB in UE.In the high-dose group, the change in mean MD from before treatment to 12 months after treatment was from − 23.67 ± 10.96 dB to −20.29 ± 10.61 dB in IE, an improvement of 3.38 dB (MD > 3 dB), meeting the criteria for improvement defined in this trial, and from − 21.39 ± 11.23 dB to −20.17 ± 11.65 dB in UE.However, there was no statistically significant difference (p > 0.05) in the MD of both groups compared to baseline at different follow-up time points after treatment.
OCT
Different doses of gene therapy had no statistically significant effect on mean RNFL thickness of IE and UE between the two groups (Fig. 2G, H).In the low-dose group, the change in mean RNFL thickness from baseline to 12 months after treatment was from 39.79 ± 5.17 μm to 41.75 ± 5.77 μm in IE and from 43.46 ± 11.24 μm to 46.17 ± 9.4 μm in UE.In the high-dose group, the change in mean RNFL thickness from before treatment to 12 months after treatment was from 47.67 ± 10.42 μm to 47.58 ± 9.60 μm in IE and from 50.83 ± 8.31 μm to 48.29 ± 8.61 μm in UE.There was no statistically significant difference (p > 0.05) in the RNFL thickness of both groups compared to baseline at different follow-up time points after treatment.
Discussion
Intraocular inflammation has been reported as one of the most common side effects in clinical studies of AAV-mediated ocular gene therapy [15, 16]. To prevent the possible immune sequence response that adeno-associated viruses 2-mediated adverse events, during and after treatment, the 12 patients were placed on a 3-week course of oral prednisone.There was no serious AEs or complications associated with the study drug in either group.This indicates that gene therapy is safe at both doses, which is consistent with previous clinical study [12].
The 10 cases of m.3460G > A mutation and 2 cases of m.3700G > A mutation included in the present study had alanine residues switched to threonine residues at positions 52 and 132 of the ND1 gene, respectively [14, 17]. Among them, m.3460G > A is the principal site of MT-ND1 [4]. Both mutations are found in the ND1 gene sequence.The drug we used is a synthetic, optimized, complete ND1 nucleotide sequence packaged in an adeno-associated virus vector, creating a drug for gene therapy.This can be used for the treatment of LHON, which is caused by different mutation sites in the ND1 subunit, to induce the expression of ND1 protein at the site of disease.The mechanism of ND1 gene therapy for LHON is similar to that for ND4,and we hypothesize that the treatment of the ND1 gene mutation will yield vision recovery similar to the treatment of ND4 gene mutation.
In this study, we also found unilateral gene therapy to improve bilateral vision.The results have been confirmed by several studies related to LHON caused by ND4 mutation [7–12, 18]. However, the mechanism of bilateral visual improvement with unilateral gene therapy remains unclear .In a primate study, unilateral intravitreal injection of rAAV2/2-ND4,quantifiable viral vector DNA was detected in the anterior segment, retina, and optic nerve of the uninjected eye, indicating that the rAAV2/2-ND4 drug was expressed in the contralateral eye [19]. There are several hypotheses for the interocular transfer of rAAV2/2-ND4 virus vector.Yang S et al. [8] found that unilateral intravitreal injection of fluorogold, the fluorogold was detected in the contralateral uninjected eye, suggesting a possible connection through axoplasmic transport.Yin et al. [20] found that it can activates macrophages to release pro-inflammatory substances by intraocular injection and promoted the regeneration of the optic nerve axons.In addition, the interconnectivity of astrocytes allows for long-distance intercellular transfer of cytoplasmic elements via membrane junctions [21]. Other scholars believe that the improvement of BCVA in the uninjected eyes is related to brain remodeling, environmental, psychological factors [22].
In the injected eyes, the visual improved by 0.22 logMAR at 12 months after treatment in the low-dose group, the visual improved only by 0.05 logMAR in the high-dose group.Whereas, in the high-dose group, the uninjected eyes visual improved by 0.22 logMAR from baseline.The REVEAL Study also shows similar results.Visual improvement in untreated eyes was more significant in the high-dose group(1.8 × 1011 vg/eye) than in the treated eyes during the 5-year follow-up.Moreover, the improvement of visual acuity in the high-dose group was less than that in the medium-high dose group(3 × 1010 vg/eye,9 × 1010 vg/eye)20.The reason is not clear.Therefore, more clinical studies are needed to explore in the future.
The results of the present study show that there were no significant differences in binocular VF (VFI, MD) and RNFL thickness between the two groups of patients 12 months after treatment when compared to the time before treatment, indicating that the changes in VF and RNFL thickness after gene therapy did not change along with BCVA and that there was no clear correlation between them or with the dosing.Although the visual field indicators and RNFL thickness did not improve significantly, they also did not continue to deteriorate and were maintained at a relatively stable state.In contrast, previous studies have found a continuing downward tendency in RNFL thickness in patients during the natural disease course of LHON [23, 24], indicating that the gene drug may have had a protective effect on the optic nerve.
This study has not been able to prove the optimal drug titer, and future studies need to increase drug safety detection indicators. To further verify these findings, a long-term, large sample, multi-center and randomized controlled studies is necessary.
Acknowledgements
The authors thank the patients and their family members for accepting gene therapy and for participating in this research.
Funding
This study was funded by Wuhan Science and Technology Major Project (grant number 2021022002023425).
Declarations
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board of Shiyan Taihe Hospital, Hubei University of Medicine (approval no.:201919) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Competing interest
All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Xin Li and Jun Yuan contributed equally to this work.
Contributor Information
Yanping Dan, Email: Danyanpin666@163.com.
Yong Zhang, Email: inforzy@163.com.
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