Molecular Therapy. Methods & Clinical Development最新文献

{"title":"A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice.","authors":"Lifang Zhou,&nbsp;Jing Su,&nbsp;Jie Long,&nbsp;Rui Tao,&nbsp;Wenling Tang,&nbsp;Fengming Qin,&nbsp;Nan Liu,&nbsp;Yanhong Wang,&nbsp;Yaoge Jiao,&nbsp;Yun Hu,&nbsp;Lurong Jiang,&nbsp;Li Li,&nbsp;Yang Yang,&nbsp;Shaohua Yao","doi":"10.1016/j.omtm.2022.01.001","DOIUrl":"https://doi.org/10.1016/j.omtm.2022.01.001","url":null,"abstract":"<p><p>Base editing tools enabled efficient conversion of C:G or A:T base pairs to T:A or G:C, which are especially powerful for targeting monogenic lesions. However, <i>in vivo</i> correction of disease-causing mutations is still less efficient because of the large size of base editors. Here, we designed a dual adeno-associated virus (AAV) strategy for <i>in vivo</i> delivery of base editors, in which deaminases were linked to Cas9 through the interaction of GCN4 peptide and its single chain variable fragment (scFv) antibody. We found that one or two copies of GCN4 peptide were enough for the assembly of base editors and produced robust targeted editing. By optimization of single-guide RNAs (sgRNAs) that target phenylketonuria (PKU) mutation, we were able to achieve up to 27.7% correction <i>in vitro</i>. <i>In vivo</i> delivery of this dual AAV base editing system resulted in efficient correction of PKU-related mutation in neonatal mice and subsequent rescue of hyperphenylalaninemia-associated syndromes. Considering the similarity between Cas9 proteins from different organisms, our delivery strategy will be compatible with other Cas9-derived base editors.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"230-240"},"PeriodicalIF":4.7,"publicationDate":"2022-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8803597/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39905549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Confirmatory detection of neutralizing antibodies to AAV gene therapy using a cell-based transduction inhibition assay.","authors":"Theresa Kasprzyk,&nbsp;Sabrina Triffault,&nbsp;Brian R Long,&nbsp;Stephen J Zoog,&nbsp;Christian Vettermann","doi":"10.1016/j.omtm.2022.01.004","DOIUrl":"https://doi.org/10.1016/j.omtm.2022.01.004","url":null,"abstract":"<p><p>Successful treatment with adeno-associated virus (AAV)-based gene therapies can be limited by pre-existing anti-AAV antibodies. Cell-based transduction inhibition (TI) assays are useful to characterize the neutralizing potential of anti-AAV antibodies in patient samples. While these assays are commonly used, they are not specific for neutralizing antibodies (NAbs) against AAV, also detecting non-antibody-based factors that inhibit AAV transduction <i>in vitro</i> but may not substantially decrease efficacy <i>in vivo</i>. This paper describes the development and bioanalytical validation of a confirmatory assay to improve the specificity of detecting anti-AAV5 NAbs in cell-based TI assays. Samples that screen positive for transduction inhibitors are subsequently depleted of all classes of immunoglobulins using agarose resins conjugated with protein A, G, and L (AGL), which restores AAV5 transduction for NAb-containing samples. Unconjugated agarose resin serves as a mock control for non-specific depletion effects and facilitates normalization of the transduction efficiencies between an AGL- and mock-treated sample; the normalized value is termed the AGL/mock ratio. During validation, a confirmatory cut point for the AGL/mock ratio was derived; sensitivity, precision, selectivity, and matrix interference were also assessed. This confirmatory TI assay facilitates a characterization of humoral immunity to AAV gene therapy by reliably distinguishing NAbs from non-antibody-based neutralizing factors.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"222-229"},"PeriodicalIF":4.7,"publicationDate":"2022-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f0/69/main.PMC8803586.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39905548","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Consensus guidelines for the validation of qRT-PCR assays in clinical research by the CardioRNA consortium.","authors":"David de Gonzalo-Calvo,&nbsp;Monica Marchese,&nbsp;Jan Hellemans,&nbsp;Fay Betsou,&nbsp;Nanna Lond Skov Frisk,&nbsp;Louise Torp Dalgaard,&nbsp;Päivi Lakkisto,&nbsp;Carole Foy,&nbsp;Andreas Scherer,&nbsp;María Laura Garcia Bermejo,&nbsp;Yvan Devaux","doi":"10.1016/j.omtm.2021.12.007","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.12.007","url":null,"abstract":"<p><p>Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking<i>.</i> Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and <i>in vitro</i> diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www.cardiorna.eu). Their applicability encompasses all clinical areas.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"171-180"},"PeriodicalIF":4.7,"publicationDate":"2022-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cc/0f/main.PMC8792405.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39590515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences.","authors":"Prasad D Trivedi,&nbsp;Chenghui Yu,&nbsp;Payel Chaudhuri,&nbsp;Evan J Johnson,&nbsp;Tina Caton,&nbsp;Laura Adamson,&nbsp;Barry J Byrne,&nbsp;Nicole K Paulk,&nbsp;Nathalie Clément","doi":"10.1016/j.omtm.2021.12.006","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.12.006","url":null,"abstract":"<p><p>Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"154-170"},"PeriodicalIF":4.7,"publicationDate":"2021-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/5e/a9/main.PMC8760416.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39943742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accelerating clinical-scale production of BCMA CAR T cells with defined maturation stages.","authors":"Jara J Joedicke,&nbsp;Ulrich Großkinsky,&nbsp;Kerstin Gerlach,&nbsp;Annette Künkele,&nbsp;Uta E Höpken,&nbsp;Armin Rehm","doi":"10.1016/j.omtm.2021.12.005","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.12.005","url":null,"abstract":"<p><p>The advent of CAR T cells targeting CD19 or BCMA on B cell neoplasm demonstrated remarkable efficacy, but rapid relapses and primary refractoriness remains challenging. A leading cause of CAR T cell failure is their lack of expansion and limited persistence. Long-lived, self-renewing multipotent T memory stem cells (T_SCM) and T central memory cells (T_CM) likely sustain superior tumor regression, but their low frequencies in blood from cancer patients impose a major hurdle for clinical CAR T production. We designed a clinically compliant protocol for generating BCMA CAR T cells starting with increased T_SCM/T_CM cell input. A CliniMACS Prodigy process was combined with flow cytometry-based enrichment of CD62L⁺CD95⁺ T cells. Although starting with only 15% of standard T cell input, the selected T_SCM/T_CM material was efficiently activated and transduced with a BCMA CAR-encoding retrovirus. Cultivation in the presence of IL-7/IL-15 enabled the harvest of CAR T cells containing an increased CD4⁺ T_SCM fraction and 70% T_SCM cells amongst CD8⁺. Strong cell proliferation yielded cell numbers sufficient for clinical application, while effector functions were maintained. Together, adaptation of a standard CliniMACS Prodigy protocol to low input numbers resulted in efficient retroviral transduction with a high CAR T cell yield.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"181-198"},"PeriodicalIF":4.7,"publicationDate":"2021-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/70/a6/main.PMC8791860.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39590516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers.","authors":"Choong-Ryoul Sihn,&nbsp;Britta Handyside,&nbsp;Su Liu,&nbsp;Lening Zhang,&nbsp;Ryan Murphy,&nbsp;Bridget Yates,&nbsp;Lin Xie,&nbsp;Richard Torres,&nbsp;Chris B Russell,&nbsp;Charles A O'Neill,&nbsp;Erno Pungor,&nbsp;Stuart Bunting,&nbsp;Sylvia Fong","doi":"10.1016/j.omtm.2021.12.004","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.12.004","url":null,"abstract":"<p><p>Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as a treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissues. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and non-human primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in a predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and non-human primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"142-153"},"PeriodicalIF":4.7,"publicationDate":"2021-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/83/f8/main.PMC8749450.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunological evaluation of an inactivated SARS-CoV-2 vaccine in rhesus macaques.","authors":"Hongbo Chen,&nbsp;Zhongping Xie,&nbsp;Runxiang Long,&nbsp;Shengtao Fan,&nbsp;Heng Li,&nbsp;Zhanlong He,&nbsp;Kangwei Xu,&nbsp;Yun Liao,&nbsp;Lichun Wang,&nbsp;Ying Zhang,&nbsp;Xueqi Li,&nbsp;Xingqi Dong,&nbsp;Tangwei Mou,&nbsp;Xiaofang Zhou,&nbsp;Yaoyun Yang,&nbsp;Lei Guo,&nbsp;Jianbo Yang,&nbsp;Huiwen Zheng,&nbsp;Xingli Xu,&nbsp;Jing Li,&nbsp;Yan Liang,&nbsp;Dandan Li,&nbsp;Zhimei Zhao,&nbsp;Chao Hong,&nbsp;Heng Zhao,&nbsp;Guorun Jiang,&nbsp;Yanchun Che,&nbsp;Fengmei Yang,&nbsp;Yunguang Hu,&nbsp;Xi Wang,&nbsp;Jing Pu,&nbsp;Kaili Ma,&nbsp;Lin Wang,&nbsp;Chen Cheng,&nbsp;Weiguo Duan,&nbsp;Dong Shen,&nbsp;Hongling Zhao,&nbsp;Ruiju Jiang,&nbsp;Xinqiang Deng,&nbsp;Yan Li,&nbsp;Hailian Zhu,&nbsp;Jian Zhou,&nbsp;Li Yu,&nbsp;Mingjue Xu,&nbsp;Huijuan Yang,&nbsp;Li Yi,&nbsp;Zhenxin Zhou,&nbsp;Jiafang Yang,&nbsp;Nan Duan,&nbsp;Huan Yang,&nbsp;Wangli Zhao,&nbsp;Wei Yang,&nbsp;Changgui Li,&nbsp;Longding Liu,&nbsp;Qihan Li","doi":"10.1016/j.omtm.2021.08.005","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.08.005","url":null,"abstract":"<p><p>Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"108-118"},"PeriodicalIF":4.7,"publicationDate":"2021-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fe/5e/main.PMC8387130.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39385312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comprehensive preclinical study supporting clinical trial of oncolytic chimeric poxvirus CF33-hNIS-anti-PD-L1 to treat breast cancer.","authors":"Shyambabu Chaurasiya,&nbsp;Annie Yang,&nbsp;Zhifang Zhang,&nbsp;Jianming Lu,&nbsp;Hannah Valencia,&nbsp;Sang-In Kim,&nbsp;Yanghee Woo,&nbsp;Suanne G Warner,&nbsp;Tove Olafsen,&nbsp;Yuqi Zhao,&nbsp;Xiwei Wu,&nbsp;Seymour Fein,&nbsp;Linda Cheng,&nbsp;Maria Cheng,&nbsp;Nicholas Ede,&nbsp;Yuman Fong","doi":"10.1016/j.omtm.2021.12.002","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.12.002","url":null,"abstract":"<p><p>CF33-hNIS-anti-PD-L1 is an oncolytic chimeric poxvirus encoding two transgenes: human sodium iodide symporter and a single-chain variable fragment against PD-L1. Comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics and biodistribution and safety studies were performed to support the clinical development of CF33-hNIS-anti-PD-L1. Most of the studies were performed in triple-negative breast cancer (TNBC) models, as the phase I trial is planned for patients with TNBC. Biological functions of virus-encoded transgenes were confirmed, and the virus demonstrated anti-tumor efficacy against TNBC models in mice. In a good laboratory practice (GLP) toxicology study, the virus did not produce any observable adverse effects in mice, suggesting that the doses proposed for the clinical trial should be well tolerated in patients. Furthermore, no neurotoxic effects in mice were seen following intracranial injection of the virus. Also, the risk for horizontal transmission of CF33-hNIS-anti-PD-L1 was assessed in mice, and our results suggest that the virus is unlikely to transmit from infected patients to healthy individuals. Finally, the in-use stability and compatibility of CF33-hNIS-anti-PD-L1 tested under different conditions mimicking the clinical scenarios confirmed the suitability of the virus in clinical settings. The results of these preclinical studies support the use of CF33-hNIS-anti-PD-L1 in a first-in-human trial in patients with TNBC.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"102-116"},"PeriodicalIF":4.7,"publicationDate":"2021-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8718831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39678510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum: Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.","authors":"Salvatore Pasqua,&nbsp;Maria Concetta Niotta,&nbsp;Giuseppina Di Martino,&nbsp;Davide Sottile,&nbsp;Bruno Douradinha,&nbsp;Monica Miele,&nbsp;Francesca Timoneri,&nbsp;Mariangela Di Bella,&nbsp;Nicola Cuscino,&nbsp;Chiara Di Bartolo,&nbsp;Pier Giulio Conaldi,&nbsp;Danilo D'Apolito","doi":"10.1016/j.omtm.2021.11.001","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.11.001","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1016/j.omtm.2021.05.002.].</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"567-568"},"PeriodicalIF":4.7,"publicationDate":"2021-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8606327/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39683346","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetically edited hepatic cells expressing the NTCP-S267F variant are resistant to hepatitis B virus infection.","authors":"Takuro Uchida,&nbsp;Seung Bum Park,&nbsp;Tadashi Inuzuka,&nbsp;Min Zhang,&nbsp;Joselyn N Allen,&nbsp;Kazuaki Chayama,&nbsp;T Jake Liang","doi":"10.1016/j.omtm.2021.11.002","DOIUrl":"https://doi.org/10.1016/j.omtm.2021.11.002","url":null,"abstract":"<p><p>The sodium-dependent taurocholate co-transporting polypeptide (NTCP)-S267F variant is known to be associated with a reduced risk of hepatitis B virus (HBV) infection and disease progression. The NTCP-S267F variant displays diminished function in mediating HBV entry, but its function in HBV infection has not been fully established in more biologically relevant models. We introduced the NTCP-S267F variant and tested infectivity by HBV in genetically edited hepatic cells. HepG2-NTCP clones with both homozygous and heterozygous variants were identified after CRISPR base editing. NTCP-S267F homozygous clones did not support HBV infection. The heterozygote clones behaved similarly to wild-type clones. We generated genetically edited human stem cells with the NTCP-S267F variant, which differentiated equally well as wild-type into hepatocyte-like cells (HLCs) expressing high levels of hepatocyte differentiation markers. We confirmed that HLCs with homozygous variant did not support HBV infection, and heterozygous variant clones were infected with HBV equally as well as the wild-type cells. In conclusion, we successfully introduced the S267F variant by CRISPR base editing into the NTCP/SLC10A gene of hepatocytes, and showed that the variant is a loss-of-function mutation. This technology of studying genetic variants and their pathogenesis in a natural context is potentially valuable for therapeutic intervention against HBV.</p>","PeriodicalId":517056,"journal":{"name":"Molecular Therapy. Methods & Clinical Development","volume":" ","pages":"597-605"},"PeriodicalIF":4.7,"publicationDate":"2021-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/8d/83/main.PMC8608598.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39683349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}