Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Lombardo, Angelo; Genovese, Pietro; Beauséjour, Christian; Colleoni, Silvia; Lee, Ya Li; Kim, Kenneth; Ando, Dale; Urnov, Fyodor D.; Galli, Cesare; Gregory, Philip D.; Holmes, Michael C.; Naldini, Luigi

doi:10.1038/nbt1353

Cited by 770 publications

(426 citation statements)

References 53 publications

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“…Meganucleases that combine the power of sequence-specific DNA binding with the ability to generate double-stranded breaks has emerged as an enabling technology of genome editing in human cells. They include zinc finger nuclease (50,53,54), TALENs (55)(56)(57)(58)(59), and the CRISPR-Cas9 system (60,61). We used TALEN technology to generate CIDEB-knockout Huh-7.5 cells and confirmed the inhibition of HCV infection and the lack of a significant effect on WNV or VSV infections in these cells.…”

Section: Discussionmentioning

confidence: 99%

Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes

Lee

Hammack

et al. 2014

J Virol

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The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.V iruses depend on host factors to gain entry into host cells, and the interaction between viral glycoproteins and cellular entry factors is important for this process and contributes to viral tropism. Of the two glycoproteins (E1 and E2) encoded by hepatitis C virus (HCV), E2 is a major target for neutralizing antibodies with well-defined epitopes, both linear and conformational (reviewed in reference 1); two of the HCV receptors, CD81 and scavenger receptor BI (SRB1), were identified through direct interaction with E2 (2, 3), and the crystal structure of a core domain of E2 has been recently solved (4). The structure and function of E1 are less well understood, but it may facilitate the correct folding (5, 6) and receptor binding (7) of E2. It has also been reported to interact with cell surface proteins (8, 9).Following attachment and receptor binding, HCV enters the cell via endocytosis with the help of additional entry cofactors (10-14). Details of the membrane fusion process of HCV entry remain poorly defined. Both the E1 and E2 proteins contain putative fusion peptides (15-17) and may participate in membrane fusion, and the crystal structure of HCV E2 suggests that HCV glycoproteins may use a fusion mechanism that is distinct from that of related positive-strand RNA viruses, includ...

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Section: Discussionmentioning

confidence: 99%

Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes

Lee

Hammack

et al. 2014

J Virol

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“…(integration-deficient lentiviruses 13 will be tested in future studies). Ex vivo delivery of the AAV6-donor with Z3 nec-mRNA-NP resulted in successful HDR in primary fibroblasts (Supplementary Fig.…”

Section: Sp-b Deficiencymentioning

confidence: 99%

In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency

et al. 2015

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In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency (2015) Nature Biotechnology, 33 (6), pp. 584-586.In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiencyTo the Editor:Nuclease-mediated genome editing holds great potential to knock out or repair diseasecausing genes. An ideal nuclease delivery vehicle is short-lived, does not integrate into the genome, and can enter target cells efficiently. These requirements have not yet been achieved simultaneously by any nuclease delivery vector. We and others have used modified mRNA, which is non-integrating and provides a transient pulse of protein expression, as an alternative to traditional viral vectors [1][2][3][4][5] . This approach allowed us to deliver therapeutic proteins in mouse models of Surfactant Protein B (SP-B) deficiency 3 and experimental asthma 4 . Here we apply it to deliver site-specific nucleases, demonstrating the value of nuclease-encoding chemically modified (nec) mRNA as a tool for in vivo genome editing. We chose a well-established transgenic mouse model of SP-B deficiency 6 in which SP-B cDNA is under the control of a tetracycline-inducible promoter 7 . Administration of doxycycline drives SP-B expression levels similar to those in wild-type mice (Supplementary Fig. 1), whereas cessation of doxycycline leads to phenotypic changes similar to those of the human disease, including thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, augmented cytokines in the lavage, a decline in lung function, and fatal respiratory distress leading to death within days 8,9 . We inserted a constitutive CAG promoter immediately upstream of the SP-B cDNA to allow doxycycline-independent expression and prolonged life in treated mice.First, we customized a panel of ZFNs and TALENs targeting the transgenic SP-B cassette ( Fig. 1a and Supplementary Fig. 2). We chose TALEN #1 (T1) and ZFN #3 (Z3) owing to their high activity and proximity to the desired site of promoter integration (Figs. 1a,b; amino acid sequences in Supplementary Fig. 4) and compared delivery by plasmid 1 DNA and mRNA. mRNA delivery resulted in higher levels of double-strand break (DSB)-induction ( Fig. 1c and Supplementary Fig. 3; P < 0.05) and homology-directed repair (HDR) ( Fig. 1d, P < 0.05). As Z3 mRNA was more efficient than T1 mRNA in both cases, Z3 was chosen for further experimentation. Comparison with a Z3-encoding AAV serotype 6 vector (AAV6) ("Z3 AAV") shows the relatively transient expression of Z3 mRNA (Fig. 1e), limiting the time during which off-target cleavage activity could occur.To optimize Z3 expression in the mouse lung, we administered a panel of 3xFLAG-tagged Z3 mRNAs with various modification schemes 2,5,10 , with or without complexation to biocompatible, biodegradable nanoparticles (NPs) made of chitosan-coated poly (lactic-coglycolic) acid (chit-PLGA) 11,12 . Following intratracheal (i.t.) delivery, NP-complexation significantly increased mRNA expression levels ( Supplem...

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“…Their ability to bind to their target DNA sites in vitro was determined using an ELISA-based assay as previously described [3]. ZFNs targeting intron 2 of the bovine CSN2 gene used in this study are available through Sigma-Aldrich (St. Louis, MO; lot number: 08181029MN).…”

Section: Materials and Methods (A) Zinc-finger Nuclease And Donor Dna mentioning

confidence: 99%

“…Zinc-finger nuclease (ZFN) technology has enabled efficient, site-specific gene modification in live cells [1][2][3][4]. ZFNs can be engineered to introduce a doublestrand break (DSB) at a pre-determined site in the genome by combining the non-specific nuclease domain of the type IIS restriction enzyme FokI with an array of zinc-finger domains that bind in the major groove of DNA and recognize a desired DNA target site [5].…”

Section: Introductionmentioning

confidence: 99%

Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases

et al. 2014

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ResearchCite this article: Liu X et al. Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed doublestrand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine b-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in b-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

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Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Cited by 770 publications

References 53 publications

Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes

Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes

In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency

Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases

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