Eric Ehrke-Schulz

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.

A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration

For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

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.

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.

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.

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.

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

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.

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.

15. A Novel Fast Production Pipeline for High Capacity Adenoviral Vectors to Deliver All Components of CRISPR/Cas9 System for Somatic Gene Editing Using One Single Viral Vector with Multiple Guide RNAs

The discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system changed the field of in vivo genome editing. Nevertheless viral delivery of all required components including Cas9 and one or multiple guide RNA (gRNA) expression units using one single viral vector has not been fully exploited. Gene deleted high-capacity adenoviral vectors (HCAdVs) have the potential to efficiently deliver all expression units of the complete CRISPR/Cas9 machinery including multiple gRNAs into a broad variety of target cells using a single viral vector. However, the complicated handling of large DNA constructs and the time consuming production procedure hampered the use of HCAdV to deliver the CRISPR/Cas9 machinery for genome editing approaches. This work aimed at adapting a toolbox for HCAdV genome manipulation for the fast and simple introduction of the customized CRISPR/Cas9 machinery to provide new instruments to improve somatic genome editing approaches in mammalian cells. We generated a new CRISPR/Cas9 shuttle plasmid toolbox containing the Cas9 nuclease gene either utilizing a constitutive or an inducible promotor and a gRNA expression unit enabling customizing the CRISPR/Cas9 for a desired target sequence. This allows cloning or recombining all CRISPR/Cas9 components into the HCAdV genome in one step. To use several gRNA expression units for multiplexing the CRISPR/Cas9 system, further gRNA expression units can be easily included. To enable fast assembly of recombinant CRISPR-HCAdV genomes we used DNA recombineering to introduce all CRISPR/Cas9 expression units into the HCAdV genome contained in the bacterial artificial chromosome pBHCA. For insertion of multiple gRNA expression units into the HCAdV genome we utilized the established pAdV-FTC plasmid in concert with homing endonuclease directed cloning. CRISPR-HCAdVs were produced using a shortened amplification and purification procedure. Exploiting our toolbox we produced several CRISPR-HCAdVs carrying single and multiplex gRNA units specific for different targets including hCCR5, hDMD, and HPV16- and HPV18-E6 genes yielding sufficient titers within a short time. T7E1 assays were applied to prove CRISPR/Cas9-mediated cleavage of respective targets and infection of cultured human cells with respective CRISPR-HCAdVs resulted in efficient site specific gene editing. In summary, this new platform enables customization, cloning and production of CRISPR-HCAdV vectors for single or multiplex approaches within a short time. It simplifies the delivery of the CRISPR/Cas9 machinery by only using one single viral vector. Inducible Cas9 expression helps to avoid targeting of the genome of producer cell lines during vector production and may be beneficial for special approaches where constitutive expression is unwanted. We speculate that this may pave the way for broader applications of the CRISPR technology in preclinical and eventually clinical studies.

104. A Gene Deleted High Capacity Adenoviral Vector for Efficient Delivery of a Multiplex DMD Specific CRISPR/Cas9 Machinery

Recent advances in the field of designer nuclease directed genome editing hold great promise to correct underlying mutations leading to Duchenne muscular dystrophy. Especially the CRISPR/Cas9 system provides an easy way to design and to assemble RNA guided nucleases offering the potential to develop personalized treatments to correct the multiple different mutations leading to this fatal disease. Recent studies showed efficient genome editing in a myoblast cell line derived from DMD patients and mdx mice. Nevertheless viral delivery of all required CRISPR/Cas9 components including Cas9 and one or multiple guide RNA (gRNA) expression units has not been fully exploited. Gene deleted high-capacity adenoviral vectors (HCAdVs) offer the packaging capacity to deliver the complete CRISPR/Cas9 machinery including several gRNA expression units using a single viral vector. By using a new toolbox that facilitates customization, cloning and production of CRISPR-HCAdVs, we assembled a HCAdV genome containing a Streptococcus pyogenes Cas9 (spCas9) gene including two guide RNA (gRNA) expression units specific for DMD that have shown efficiency to delete exon 51 in dystrophic human myoblasts. CRISPR-HCAdV was amplified in medium scale using a shortened protocol yielding high titers. Infection of cultured HEK293 cells and primary human myoblasts with purified DMD specific CRISPR-HCAdV at different MOIs resulted in strong locus specific deletion efficiency for DMD exon 51 as shown with locus specific PCR. As a comparison we also designed and produced DMD-specific TALEN encoding HCAdVs which allow delivery of a complete TALEN pair using a single vector. We found that it was more complicated to produce double TALEN-HCAdVs compared to multiplex CRSIPR/Cas9-HCAdV as they require controlled expression of TALEN genes by inducible promotors. Furthermore the TALEN system is not suitable for multiplexing and showed less efficiency in T7E1 assays. Our platform enables cloning and production of gene deleted adenoviral vectors for the delivery of a DMD specific CRISPR/Cas9 system within a short time providing a valuable tool for viral delivery of customized CRISPR/Cas9 for DMD treatment. Additional gRNAs or gRNAs with other specificities can be easily included in the vector allowing personalized molecular design of the gene transfer vector. We expect that this may pave the way towards broader applications of the CRISPR technology for DMD treatment including preclinical and eventually clinical studies.