ABSTRACT
Objective:To evaluate the safety and clinical effect of gene therapy for Leber hereditary optic neuropathy (LHON).Methods:A multi-center prospective non-randomized controlled trial was conducted.Eighty eyes of 40 LHON patients with mitochondrial DNA 11778 mutation were enrolled in Taihe Hospital from December 2017 to February 2018.Intravitreal injection of recombinant adeno associated virus 2-NADH dehydrogenase 4 (rAAV2- ND4) was carried out in the unilateral eye with worse visual acuity or the right eye (if the visual acuity of both eyes was equal) of each subject as the treated group and the fellow eyes as the untreated group.The best corrected visual acuity (BCVA) was detected using a standard logarithmic chart and intraocular pressure (IOP) was measured with a non-contact tonometer before treatment and 1, 3, 6, 12 months after treatment.The manifestations of the ocular anterior segment and fundus were examined by slit lamp microscopy and color photography.The changes of visual acuity and IOP before and after gene therapy were compared, and complications were evaluated between the treated group and the untreated group.The effective rate defined as visual acuity improved ≥0.3 LogMAR at the end of follow-up was assessed.This study adhered to the Declaration of Helsinki and the study protocol was approved by an Ethics Committee of Taihe Hospital (No.201807). Written informed consent was obtained from each subject prior to any medical examination and treatment. Results:The visual acuity improved 6 eyes in the treated group and 4 eyes in the untreated group, and 13 patients showed bilateral improvement.The visual acuity improvement ≥0.3 LogMAR in 23 patients with the effective rate 57.5%.The BCVA was (1.51±0.62) LogMAR and (1.62±0.58) LogMAR at the end of following-up in the untreated group and treated group, respectively, which were significantly higher than (1.75±0.46) LogMAR and (1.83±0.47) LogMAR before treatment (both at P<0.01), and no significant difference was found in BCVA between the two groups ( Fgroup=0.084, P=0.772). There was no significant difference in IOP between the two groups before and after treatment ( Fgroup=0.557, P=0.575; Ftime=2.314, P=0.106). No serious complications were found in all subjects during following-up. Conclusions:rAAV2- ND4 gene therapy is safe and effective for LHON, and binocular vision can be improved by monocular intravitreal injection of rAAV2- ND4 gene.
ABSTRACT
Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.