Determination of pre-existing levels of adeno-associated virus-specific neutralizing antibodies in the vitreous and. We determine the prevalence of neutralizing antibodies (NAbs) against adeno-associated virus (AAV) in vitreous and serum of patients with vitreoretinal diseases and investigate the relationship between NAb titers in vitreous and serum. NAb levels were much lower in vitreous than in serum, regardless of serotype.
Patients with VH had higher levels of NAbs in the vitreous than those without VH. Accordingly, a correlation between serum and vitreous NAb titers has also not been established to our knowledge. To our knowledge, we report the first analytical screening of NAbs against four different AAV serotypes (2, 5, 8 and 9) in the vitreous and serum of patients with various vitreoretinal diseases.
Materials and Methods
Vitreous samples were collected in the operating room through undiluted lines with a vitreous cutter. Undiluted vitreous samples were obtained with a 3-mL syringe connected directly to the handpiece until the eye was visibly softened. At least 2 ml of undiluted vitreous was collected in each case; 3 ml serum samples were collected on the day before or on the day of surgery.
Samples were kept in the collection syringe and delivered to the clinical laboratory immediately after collection.
The results of NAb titers against each serotype in serum are shown in Table 1 and Figure 2. Similar to the results for serum, it was confirmed that the vitreous NAb titer against serotype 2 was the highest and the NAb titer against serotype 8 was the lowest. However, the absolute value of the NAb titer in the vitreous was much lower than that in the serum.
Among the underlying vitreoretinal disease states in the eight patients with NAb titers >100, five patients had diabetic VH, one had AMD-related VH, and one had a macular hole. Percent ranges of vitreous humor titer for samples positive for NAbs against each serotype are shown, as well as distribution by range of neutralizing titers against AAV2, AAV5, AAV8, and AAV9. Comparison of NAb titer and NAb ratio to AAV2 and AAV5 according to the presence or absence of leakage in patients with ERM.
Nineteen patients with preoperative VH or ERM with preoperative fluorescein leakage were classified as patients with disease in which the BRB was disturbed (group A), and 13 were classified as patients with disease in which the BRB was intact (group B; table 4). . Group B had eight patients with ERM without leakage, three with macular hole and two with intraocular lens dislocation. Comparison of NAb titer and NAb ratio with AAV2 and AAV5 with respect to BRB maintenance.
Overall, there were no significant correlations between serum and vitreous humor NAbs titers (Fig. 4). However, when the data were analyzed according to BRB maintenance, a strong positive correlation was found between serum NAbs titers and vitreous humor in patients with interrupted BRB (r = 0.917, P < 0.001), whereas no correlation was found significant in patients with intact BRBs (r = 0.381, P = 0.199). In most cases of intact BRB, vitreous humor NAb titers remained low, regardless of serum NAb titers.
Difference in the ratio of neutralizing activity against AAV serotype 2 in serum and vitreous according to BRB maintenance.
Patients with VH (ie, severely disrupted intrinsic BRB) had higher vitreous NAbs titers than patients with intact intrinsic BRB, regardless of their serum titers. The difference caused by pre-existing BRB-dependent immune status is clearer when considering the ratio of serum to vitreous NAb titers. Our study showed that when the BRB is intact, the level of NAbs in the vitreous remains low even when the serum level is high.
These results indicated that the level of vitreous NAbs could be higher in cases of BRB disorders and that the absolute value of vitreous NAbs could be affected by the extent of BRB disorders and the serum NAbs level. Based on our findings, these conflicting results could be explained by differences in BRB maintenance status in the target disease. However, the results of the non-human primate study were different from those of the human studies because the primate study reported that the presence of pre-existing NAb titers in the serum of healthy monkeys was strongly associated with weak transgene expression after intravitreal AAV administration . 9 It is suggested that immunological heterogeneity and differences in BRB structure between non-human primates and humans may account for the differences between the results of these studies.20.
Another factor to consider regarding the immunological aspect of retinal gene therapy is the route of administration; i.e. the subretinal or intravitreal space, since they differ slightly immunologically.17,21,22 A mouse study showed that intravitreal administration of AAV generated a humoral immune response against AAV capsid equivalent to that of systemic administration, whereas subretinal administration did not induce a humoral immune response and did not affect subsequent intravitreal or subretinal administration of AAV. 21 Other studies have shown that initial subretinal injection of AAV2 does not prevent readministration of AAV2 to the contralateral eye. 17,22 Human studies involving neovascular targeting AMD with subretinal injections of AAV2 reported that the presence of anti-AAV antibodies in the serum at baseline did not affect the outcome, in contrast to the findings of Heier et al. The significance of our study is that, to our knowledge, it is the first to measure pre-existing NAb titers in the human vitreous humor and to investigate pre-existing NAb titers in Korean populations to provide insights into AAV vector-based gene therapy . By examining patients with various vitreoretinal diseases, we demonstrated the possibility of differences in the relationship between NAb titers in the serum and vitreous humor according to BRB status, and we determined the factors that should be taken into account when performing retinal gene therapy are related to the inhibitory effect of NAbs.
Our results are important for the development of gene therapy for neovascular AMD in the future. However, baseline serum NAb levels and BRB cleavage rate should be considered when planning gene therapy for diseases associated with BRB cleavage, because NAb levels in the vitreous humor are influenced by these two factors and can be quite high. to influence. successful transduction.
Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness. A defect in the blood-retinal barrier in the region of the optic nerve head in the rabbit and monkey. Safety and durability of the effect of contralateral eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-up phase 1 trial.
2a상 무작위 임상 시험: 습성 연령 관련 황반 변성에 대한 망막하 rAAV.sFLT-1의 안전성 및 사후 분석. 본 연구의 목적은 안질환 환자의 혈청과 유리체액을 동시에 채취하여 유리체강 내 아데노 관련 바이러스에 대한 중화항체의 수준과 혈청과 유리체액의 중화항체의 상관관계를 알아보는 것이다. . . . 본 연구에서는 안질환 환자 중 유리체절제술을 시행하고자 하는 32명의 환자로부터 혈청 및 유리체액을 채취하여 중화항체법 프로토콜에 따라 분석하였다.
유리체 출혈과 조영제 누출을 분석하고 혈액망막장벽의 손상을 종합적으로 평가하였다. 전체 중화항체 수치는 바이러스 종류에 관계없이 유리체액에서 혈청보다 유의하게 낮았다. 그러나 유리체출혈이 있는 환자는 출혈이 없는 환자에 비해 유리체액 내 중화항체가 높게 나타났고, 혈청과 유리체액 간의 중화항체 비율(혈청 내 중화항체 농도/유리체액 내 중화항체 농도)은 유리체액 내 중화항체 농도가 유리체 출혈 후 증가하는 것으로 나타났다. epiretinal membrane이 있는 환자의 수는 그렇지 않은 환자보다 현저히 적었습니다.
유리체출혈과 망막앞누출은 혈액-망막장벽이 손상된 질환으로 분류할 수 있으며, 혈액-망막장벽이 손상된 환자는 유리체 내 중화항체가 증가할수록 중화항체 비율이 유의하게 낮아졌다.