RESUMEN
A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Interacciones Microbiota-Huesped/genética , Proteínas Nucleares/genética , Antígenos de Superficie/genética , Antivirales/farmacología , Línea Celular Tumoral , ADN Viral/genética , Estudio de Asociación del Genoma Completo/métodos , Células Hep G2 , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Interacciones Microbiota-Huesped/efectos de los fármacos , Humanos , Polinucleotido Adenililtransferasa/genética , Carga Viral/efectos de los fármacos , Carga Viral/genéticaRESUMEN
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
Asunto(s)
Hemoglobina Fetal/genética , Filtración/métodos , Edición Génica/métodos , Trasplante de Células Madre Hematopoyéticas , Ribonucleoproteínas/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Electroporación/métodos , Femenino , Hemoglobina Fetal/metabolismo , Filtración/economía , Filtración/instrumentación , Terapia Genética/economía , Terapia Genética/instrumentación , Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Hemoglobinopatías/genética , Hemoglobinopatías/terapia , Humanos , Membranas Artificiales , Ratones , Modelos Animales , ARN Guía de Kinetoplastida/genética , Trasplante AutólogoRESUMEN
CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.