Efficacy evaluation of intravitreal injection of rAAV2- ND4 gene for Leber hereditary optic neuropathy / 中华实验眼科杂志

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.

Construction of Pim-1 recombinant adeno-associated virus 2 vector and its infection in rat retina / 解剖学报

Objective: To construct a recombinant adeno-associated virus 2 vector (rAAV2-Pim-l) carrying rat proto-oncogene Pim-1, and to detect infected cell types and the expression of Pim-1 in rat retina infected by rAAV2-Pim-l. Methods: The pAOV-CAGMINI-EGFP-2A-MCS-3FLAG vector and Pim-1 PCR product were cleaved by Nhel enzyme. The recovered vector and target gene DNA were identified after agarose gel electrophores and linked to transformation, positive plasmid cloning and sequencing analysis. rAAV2-Pim-l expression plasmid pA0V-CAGMINI-EGFP-2A-Pim-l-3FLAG, packaging plasmid pAAV-RC and helper plasmid pHelper were co-transfected into 293 cells through using the Lipofectamine 2000, then high titer of rAAV2-Pim-l was purified. After rAAV2-Pim-l injection into rat intravitreal body, the infected retinal cell types were detected by immunofluorescence histochemistry staining; the expression of Pim-1 in the retina was quantified by Real-time PCR and Western blotting. Results: The construction of rAAV2-Pim-l plasmid was successful and the nucleotide sequence was right; Green fluorescence in 293 cells was found after plasmid transfection into 293 cells; The virus titer was 5.7 × 1015vg/L. After rAAV2-Pim-l infected rat retina in vivo, the infection percentage of retinal ganglion cells (RGCs) reached 71%, and a few of amacrine cells and hardly astrocytes were infected; the expression of Pim-1 mRNA and protein in the retina of rAAV2-Pim-l group was about 6. 61 times and 2. 29 times of the rAAV2-EGFP group respectively. Conclusion: rAAV2-Pim-l virus vector is successfully constructed and Pim-1 is overexpressed in the RGCs of infected rat retina.

Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

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.

Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.

Role of cellular FKBP52 protein in hydroxyurea treatment-mediated increase in transduction efficiency of recombinant adeno-associated virus 2 vectors / 中国药理学通报

Aim To explore the role of cytoplasmic FKBP52 in AAV-mediated transduction.Methods Murine embryo fibroblasts(MEFs)cultures from FKBP52 wild-type(WT),heterozygous(HE),and knockout(KO)mice were established.The role of FKBP52 in intracellular trafficking of AAV was analyzed by fluorescence-activated cell sorting(FACS)analyses,electrophoretic mobility shift assays(EMSA),southern blot,immunoprecipitations and western blot analyses.Results Conventional AAV vectors failed to transduce WT MEFs efficiently,and the transduction efficiency was not significantly increased in HE or KO MEFs.AAV vectors failed to traffick efficiently to the nucleus in these cells.Treatment with hydroxyurea(HU)increased the transduction efficiency of conventional AAV vectors by～25-fold in WT MEFs,but only by～4-fold in KO MEFs.The use of self-complementary AAV(scAAV)vectors,which bypass the requirement of viral second-strand DNA synthesis,revealed that HU treatment increased the transduction efficiency～23-fold in WT MEFs,but only～4-fold in KO MEFs,indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis.Following HU treatment,～59% of AAV genomes were present in the nuclear fraction from WT MEFs,but only ～28% in KO MEFs,indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs.When KO MEFs were stably transfected with an FKBP52 expression plasmid,HU treatment-mediated increased in the transduction efficiency was restored in these cells,which correlated directly with improved intracellular trafficking.Intact AAV particles were also shown to interact with FKBP52 as well as with dynein,a known cellular protein involved in AAV trafficking.Conclusion These studies suggest that FKBP52,being a cellular chaperone protein,facilitates intracellular trafficking of AAV,which has implications in the optimal use of recombinant AAV vectors in human gene therapy.