Designer nuclease-mediated gene correction via homology-directed repair in an in vitro model of canine hemophilia B.

BACKGROUND: Gene correction at specific target loci provides a powerful strategy for overcoming genetic diseases. In the present study, we aimed to use an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX (cFIX) gene. To correct the defective gene via homology-directed repair (HDR), we designed transcription-activator like effector nucleases and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double-strand breaks at the mutation site. METHODS: To generate a stable cell line containing the mutated cFIX locus, a 2-kb genomic DNA fragment derived from a hemophilia B dog was amplified and integrated utilizing the phiC31 integrase system. Designer nucleases were assembled and cloned into vectors for constitutive and inducible expression. To detect mutations, insertions and deletions, and HDR events after nuclease treatment T7E1 assays, an amplification-refractory mutation system-quantitative polymerase chain reaction and pyrosequencing were performed. RESULTS: To perform HDR correction experiments, we established a cell line carrying the mutated cFIX locus. In HDR approaches we either explored a wild-type or an optimized cFIX sequence and we found that our modified HDR cassette showed higher gene correction efficiencies of up to 6.4%. Furthermore, we compared inducible and constitutive designer nuclease expression systems and found that the inducible system resulted in comparable HDR efficiencies. CONCLUSIONS: In conclusion, the present study demonstrates the potential of this strategy for gene therapeutic approaches in vitro and in a canine model for hemophilia B.

A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Adenoviral Vectors Armed with PAPILLOMAVIRUs Oncogene Specific CRISPR/Cas9 Kill Human-Papillomavirus-Induced Cervical Cancer Cells.

Human papillomaviruses (HPV) cause malignant epithelial cancers including cervical carcinoma, non-melanoma skin and head and neck cancer. They drive tumor development through the expression of their oncoproteins E6 and E7. Designer nucleases were shown to be efficient to specifically destroy HPV16 and HPV18 oncogenes to induce cell cycle arrest and apoptosis. Here, we used high-capacity adenoviral vectors (HCAdVs) expressing the complete CRISPR/Cas9 machinery specific for HPV18-E6 or HPV16-E6. Cervical cancer cell lines SiHa and CaSki containing HPV16 and HeLa cells containing HPV18 genomes integrated into the cellular genome, as well as HPV-negative cancer cells were transduced with HPV-type-specific CRISPR-HCAdV. Upon adenoviral delivery, the expression of HPV-type-specific CRISPR/Cas9 resulted in decreased cell viability of HPV-positive cervical cancer cell lines, whereas HPV-negative cells were unaffected. Transduced cervical cancer cells showed increased apoptosis induction and decreased proliferation compared to untreated or HPV negative control cells. This suggests that HCAdV can serve as HPV-specific cancer gene therapeutic agents when armed with HPV-type-specific CRISPR/Cas9. Based on the versatility of the CRISPR/Cas9 system, we anticipate that our approach can contribute to personalized treatment options specific for the respective HPV type present in each individual tumor.

Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B.

Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5, and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the tetracycline-controlled transcriptional activator (Tet-on)-inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology-directed repair (HDR). For efficient delivery of designer nuclease and donor DNA, we produced a high-capacity adenovirus vector type 5 (HCAdV5) containing the Tet-on-inducible cFIX-specific CRISPR/Cas9 system and a single-stranded adeno-associated virus type 2 vector (ssAAV2) containing the modified donor. Moreover, we designed a single HCAdV5 delivering all components for HDR. Our amplification-refractory mutation system based on qPCR analysis (ARMS-qPCR) revealed that the single vector application in Huh7-cFIXmut cells resulted in up to 5.52% HDR efficiencies, which was superior to the two-vector strategy. Furthermore the single vector also resulted in increased phenotypic correction efficiencies assayed by ELISA. We conclude that HDR in combination with viral vector delivery holds great promise for the correction of mutated FIX in disease models.

One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors.

High-capacity adenoviral vectors (HCAdVs) devoid of all coding genes are powerful tools to deliver large DNA cargos into cells. Here HCAdVs were designed to deliver a multiplexed complete CRISPR/Cas9 nuclease system or a complete pair of transcription activator-like effector nucleases (TALENs) directed against the hepatitis B virus (HBV) genome. HBV, which remains a serious global health burden, forms covalently closed circular DNA (cccDNA) as a persistent DNA species in infected cells. This cccDNA promotes the chronic carrier status, and it represents a major hurdle in the treatment of chronic HBV infection. To date, only one study demonstrated viral delivery of a CRISPR/Cas9 system and a single guide RNA (gRNA) directed against HBV by adeno-associated viral (AAV) vectors. The advancement of this study is the co-delivery of multiple gRNA expression cassettes along with the Cas9 expression cassette in one HCAdV. Treatment of HBV infection models resulted in a significant reduction of HBV antigen production and the introduction of mutations into the HBV genome. In the transduction experiments, the HBV genome, including the HBV cccDNA, was degraded by the CRISPR/Cas9 system. In contrast, the combination of two parts of a TALEN pair in one vector could not be proven to yield an active system. In conclusion, we successfully delivered the CRISPR/Cas9 system containing three gRNAs using HCAdV, and we demonstrated its antiviral effect.

Establishment of the CRISPR/Cas9 System for Targeted Gene Disruption and Gene Tagging.

CRISPR/Cas9 RNA-guided nucleases refashioned in vivo gene editing approaches for specific gene disruption, gene correction, or gene addition. Moreover, chimeric Cas9 proteins can be applied to direct fused cis-acting effector protein domains, enzymes, or fluorescent markers to DNA to target sequences to regulate gene expression, to introduce epigenetic changes, or to fluorescently label DNA sequences of interest. Here we show how to design guide RNAs for specific DNA targeting. We provide a protocol to customize the CRISPR/Cas9 machinery encoded on commercially available plasmids and present how to test the targeting efficiency of Cas9 with a target-specific gRNA by testing mutation induction efficiency. To exemplify related applications we provide a guideline of how to apply the CRISPR/Cas9 technology for gene labeling.

CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity.

Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

Quantification of designer nuclease induced mutation rates: a direct comparison of different methods.

Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented.