An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (ß-amino ester) nanoparticles for vaginal gene therapy.

BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.

In vitro and in vivo growth inhibition of human cervical cancer cells via human papillomavirus E6/E7 mRNAs' cleavage by CRISPR/Cas13a system.

Sustained infection of high-risk human papillomavirus (HR-HPVs), especially HPV16 and HPV18, is a major cause of cervical cancer. E6 and E7 oncoproteins, encoded by the HPV genome, are critical for transformation and maintenance of malignant phenotypes of cervical cancer. Here, we used an emerging programmable clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13a system to cleave HPV 16/18 E6/E7 messenger RNAs (mRNAs). The results showed that customized CRISPR/Cas13a system effectively and specifically knocked down HPV 16/18 E6/E7 mRNAs, inducing growth inhibition and apoptosis in HPV16-positive SiHa and HPV18-positive HeLa Cell lines, but not in HPV-negative C33A cell line. Simultaneously, we detected downregulation of E6/E7 oncoproteins and upregulation of tumor suppressor P53 and RB proteins. In addition, we used subcutaneous xenograft tumor growth assays to find that the weight and volume of tumors in the SiHa-16E6CR1 group knocked down by the CRISPR/Cas13a system were significantly lower than those in the SiHa-VECTOR group lacking crRNA. Our study demonstrated that targeting HPV E6/E7 mRNAs by the CRISPR/Cas13a system may be a candidate therapeutic strategy for HPV-related cervical cancer.

Comparison of the short-term and long-term outcomes of laparoscopic hysterectomies and of abdominal hysterectomies: a case study of 4,895 patients in the Guangxi Zhuang Autonomous Region, China.

BACKGROUND: To evaluate the short-term and long-term outcomes after laparoscopic hysterectomy (LH) compared with abdominal hysterectomy (AH) in case of benign gynecological disease. METHODS: A multi-center cohort retrospective comparative study of population among 4,895 hysterectomies (3,539 LH vs.1,356 AH) between 2007 and 2013 was involved. Operative time (OT), estimated blood loss (EBL), intra-operative and post-operative complications, passing flatus; days with indwelling catheter, questionnaires covering pelvic floor functions and sexual functions were assessed. RESULTS: The EBL (174.1±157.4 vs. 263.1±183.2 cc, LH and AH groups, respectively), passing flatus (38.7±14.1 vs. 48.1±13.2 hours), days with indwelling catheter (1.5±0.6 vs. 2.2±0.8 days), use of analgesics (6.5% vs. 73.1%), intra-operative complication rate (2.4% vs. 4.1%), post-operative complication rate (2.3% vs. 5.7%), post-operative constipation (12.1% vs. 24.6%), mild and serious stress urinary incontinence (SUI) post-operative (P<0.001; P=0.014), and proportion of Female Sexual Functioning Index (FSFI) total score <26.55 post-operative (P<0.001) of the LH group were significantly less than those of AH group. There were no significant differences in OT (106.5±34.5 vs. 106.2±40.3 min) between the two groups. CONCLUSIONS: LH is a safe and efficient operation for improving patients?long-term quality of life (QoL), and LH is a cost-effectiveness procedure for treating benign gynecological disease. LH is superior to AH due to reduced EBL, reduced post-operative pain and earlier passing flatus.

[Constitutive characteristics and change trend of gynecological malignant tumors in 8009 hospitalized patients in Guangxi Zhuang Autonomous Region].

OBJECTIVE: To investigate the constitutive characteristics and the change trend of gynecologic malignant tumors in hospitalized patients in Guangxi Zhuang Autonomous Region over the recent 20 years. METHODS: Clinical data of 8009 in-patients who suffered from gynecologic malignant tumors in 23 hospitals from 1985 to 2004 in Guangxi Zhuang Autonomous Region were analyzed, with respect to the tumor types and change trend. RESULTS: (1) The leading 4 types of malignant tumors were cervical cancers, ovarian cancers, endometrial cancers, and malignant trophoblastic tumors according to the constitutive ratios of the tumors. The constitutive ratio of cervical cancer patients rose year by year, from 17.48% during the 1985-1989 period to 49.25% during the 2000-2004 period (P < 0.01). While the constitutive ratio of malignant trophoblastic tumors dropped year by year. The changes of ovarian cancer, endometrial cancer, vulvar and vaginal carcinomas, and sarcoma of the uterus were not obvious (P > 0.05). (2) The occurring age of cervical cancers became younger obviously, from > or = 60 years old dropped to < 40 years old. (3) Cervical cancers were found mainly in urban residents in the former 10 years, the constitutive ratio being 67.1%; while in the latter 10 years it gradually shifted to rural residents, accounting for 52.6% of the total gynecological tumors. (4) Patients were usually at stages II, III, IV when they visited a doctor for their diseases. Especially for ovarian cancer, malignant trophoblastic tumors, sarcoma of the uterus, these patients were in the intermediate or advanced stage when they were diagnosed, mainly because of lack of obvious symptoms. The constitutive ratio of these advanced patients was over 60%. CONCLUSIONS: We should strengthen the screening program of cervical cancer, and pay more attention to prevention and control of other gynecological reproductive organ tumors at the same time. On the other hand, we should explore better tumor markers, new methods of diagnosis and treatment to improve early diagnosis and treatment of gynecologic malignant tumors.