重组复制型溶瘤单纯疱疹病毒人细胞巨噬细胞集落刺激因子的质量研究

Quality Control Research on Recombinant Oncolytic Herpes Simplex Virus Serotype 1 Encoding Human GM-CSF Gene

目的研究建立重组复制型溶瘤单纯疱疹病毒人细胞巨噬细胞集落刺激因子(GM-CSF,HSV1/GM-CSF)的质控方法与质量标准。方法采用限制性内切酶酶切与PCR法,对GM-CSF基因、单纯疱疹病毒载体ICP 34.5、ICP6、ICP47等改造结构进行鉴定。采用A260紫外吸收法检测病毒颗粒数;pfu法测定HSV1/GM-CSF感染活性。HSV1/GM-CSF体外感染Vero细胞后,分别测定感染上清中GM-CSF表达量以及表达产物的生物学活性;测定HSV1/GM-CSF对肿瘤细胞MCF7的体外杀伤活性;HSV1/GM-CSF分别以相同MOI感染MCF7与二倍体人胚肺成纤维细胞MRC5后,分别测定细胞裂解上清中子代病毒的感染活性,并以子代病毒的感染活性比值来分析该制品在肿瘤细胞中的增殖复制能力。采用PCR法控制野生型单纯疱疹病毒等外源因子的残留。结果对重组HSV1/GM-CSG基因组的酶切、以及对所携带的GM-CSF基因、ICP 34.5、ICP6、ICP47基因改造区的PCR鉴定结果与理论值相符。病毒载体颗粒数为4.5×1010VP.mL-1,感染活性为4.3×107pfu.mL-1。HSV1/GM-CSF以MOI 0.01感染Vero细胞72 h后,ELISA检测上清中GM-CSF表达量为228 ng.mL-1,表达产物的生物学活为4.3×103U.mL-1。该基因治疗制剂对人乳腺癌肿瘤细胞MCF7体外杀伤的MOIIC50为0.08。以相同MOI感染并经pfu法检测,HSV1/GM-CSF在人乳腺癌肿瘤细胞MCF7与人胚肺成纤维二倍体细胞MRC5的增殖比值为308。未检出野生型单纯疱疹病毒。其他各项指标均符合《人基因治疗研究和制剂质量控制技术指导原则》及2005年版《中国药典》(三部)要求。结论初步建立了HSV1/GM-CSF的质控方法和质量标准,并已用于该制品的质量控制。

OBJECTIVE To establish quality control methods and specifications for oncolytic herpes simplex virus serotype 1 encoding human GM-CSF gene （ HSV-1/GM-CSF）. METHODS GM-CSF gene and modified DNA region in ICP 34. 5, ICP6 and ICP47 were identified by recombinant virus vector DNA restriction enzyme digestion map and PCR analysis. Virus particle number and infectious titer were determined by UV A26o n,, and pfu methods respectively. GM-CSF expression level and biological activity in superna- tants of Vero cell, which exposed to the HSV-1/GM-CSF in vitro were determined respectively. The anti-tumor potency of oncolytic virus was evaluated by human breast carcinoma cell MCF7 cytotoxity assay. The tumor cells targeted replication ability of recombinant virus was determined through filial generation virus pfu titer ratio between human breast carcinoma MCF-cell＇and human diploid embryonic lung fibroblast MRC5 after infected by virus with same MOI respectively. Wild type herpes simplex virus serotype 1 and other adventitious virus were controlled by PCR. RESULTS The results of restriction enzyme digestion map and PCR analysis for modified ICP 34. 5, ICP6 and ICP47 region and GM-CSF gene conformed to theoretic values. The particle number and infectious titer of the virus were 4. 5 × 10^10VP · mL^-1 and 4. 3 × 10^7 pfu·mL^-1 respectively. Vero cells were exposed to HSV-1/GM-CSF with MOI 0. 01 and cultured for 72 h. The expression level and biological activity of GM-CSF in the supernatants were 228 ng · mL^-1 and 4. 3 × 103 U · mL^-1 respectively. The MOI IC50 of the in vitro anti-tumor cytotoxity potency was O. 08. Filial generation virus TCID50 titer ratio of MCF7 tumor cell infection group to MRC5 normal cell infection control group was 308 with same infection MOI. Wild type HSV was not detected. Other quality control items complied with corresponding requirements in the guidance for quality control of human gene therapy products and Chinese pharmacopeia volume Ⅲ 2005 edition. CONCLUSION The methods and specifications have been established for the quality control of oncolytie HSV-1/GM-CSF, which can also be reference for quality control of other oneolytic viral vector products.