High-capacity Adenoviral Vectors Research Articles

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease.Key messagesAdenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting.An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons.A hybrid promoter allows preferential expression of transgenes in GABAergic neurons.Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

Application of a split-Cre system for high-capacity adenoviral vector amplification.

High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.

Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations

Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient’s neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.

Adenovirus-Mediated Inducible Expression of a PD-L1 Blocking Antibody in Combination with Macrophage Depletion Improves Survival in a Mouse Model of Peritoneal Carcinomatosis.

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.

IMATCH: an integrated modular assembly system for therapeutic combination high-capacity adenovirus gene therapy

Adenovirus-mediated combination gene therapies have shown promising results in vaccination or treating malignant and genetic diseases. Nevertheless, an efficient system for the rapid assembly and incorporation of therapeutic genes into high-capacity adenoviral vectors (HCAdVs) is still missing. In this study, we developed the iMATCH (integrated modular assembly for therapeutic combination HCAdVs) platform, which enables the generation and production of HCAdVs encoding therapeutic combinations in high quantity and purity within 3 weeks. Our modular cloning system facilitates the efficient combination of up to four expression cassettes and the rapid integration into HCAdV genomes with defined sizes. Helper viruses (HVs) and purification protocols were optimized to produce HCAdVs with distinct capsid modifications and unprecedented purity (0.1 ppm HVs). The constitution of HCAdVs, with adapters for targeting and a shield of trimerized single-chain variable fragment (scFv) for reduced liver clearance, mediated cell- and organ-specific targeting of HCAdVs. As proof of concept, we show that a single HCAdV encoding an anti PD-1 antibody, interleukin (IL)-12, and IL-2 produced all proteins, and it led to tumor regression and prolonged survival in tumor models, comparable to a mixture of single payload HCAdVs in vitro and in vivo. Therefore, the iMATCH system provides a versatile platform for the generation of high-capacity gene therapy vectors with a high potential for clinical development.

Local administration of IL-12 with an HC vector results in local and metastatic tumor control in pediatric osteosarcoma

Osteosarcoma is the most frequent and aggressive bone tumor in children and adolescents, with a long-term survival rate of 30%. Interleukin-12 (IL-12) is a potent cytokine that bridges innate and adaptive immunity, triggers antiangiogenic responses, and achieves potent antitumor effects. In this work, we evaluated the antisarcoma effect of a high-capacity adenoviral vector encoding mouse IL-12. This vector harbored a mifepristone-inducible system for controlled expression of IL-12 (High-Capacity adenoviral vector enconding the EF1α promoter [HCA-EFZP]-IL-12). We found that local administration of the vector resulted in a reduction in the tumor burden, extended overall survival, and tumor eradication. Moreover, long-term survivors exhibited immunological memory when rechallenged with the same tumor cells. Treatment with HCA-EFZP-IL-12 also resulted in a significant decrease in lung metastasis. Immunohistochemical analyses showed profound remodeling of the osteosarcoma microenvironment with decreases in angiogenesis and macrophage and myeloid cell numbers. In summary, our data underscore the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which can trigger a potent antitumor immune response in primary and metastatic pediatric osteosarcoma.

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.

High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy.

The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or “gutless”. Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. More importantly, the lack of viral coding sequences in the genomes of HC-AdVs extends the cloning capacity up to 37 Kb, and allows long-term episomal persistence of transgenes in non-dividing cells. These properties open a wide repertoire of therapeutic opportunities in the fields of gene supplementation and gene correction, which have been explored at the preclinical level over the past two decades. During this time, production methods have been optimized to obtain the yield, purity, and reliability required for clinical implementation. Better understanding of inflammatory responses and the implementation of methods to control them have increased the safety of these vectors. We will review the most significant achievements that are turning an interesting research tool into a sound vector platform, which could contribute to overcome current limitations in the gene therapy field.

High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of DMD Gene Repair Tools and Strategies in Human Cells.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents.

Danio Rerio as Model Organism for Adenoviral Vector Evaluation.

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.

Abstract 3115: High-capacity adenoviral vectors with controlled expression of interleukin 12 as a new strategy against pediatric osteosarcoma

Abstract Osteosarcoma (OS) is the most frequent and aggressive primary bone tumor in children and young adults, with a high propensity to metastasize to the lungs. Despite therapeutic efforts, the survival rate for metastatic or relapsed patients in under 30%. For this reason, we need new strategies to halt the progression of primary bone tumor and impede the metastatic stage. Interleukin-12 (IL12) is a cytokine implicated in a variety of immune activities including the stimulation of T and natural killer (NK) cells and the production of high levels of IFNγ. In addition, this process triggers an anti-angiogenic response leading to T reg inhibition and tumor death. This cytokine has shown significant antitumor activity in several animal models and clinical trials. The aim of this work was to evaluate the antisarcoma effect of a high-capacity adenovirus vector carrying the mouse IL12 (mIL12). This vector harbours a mifepristone (RU486)-inducible system for controlled expression of IL12 (HCA-EFZP-IL12) in order to improve the treatment of OS and reduced the systemic toxicity associated with IL12. The murine K7M2 OS cell line was injected intratibially in Balbc mice to induce bone tumor. HCA-EFZP-IL12 was administered intratumorally (two doses 2 days apart) in the orthotopic model with mifepristone induction during two weeks. Blood samples were collected 10 h after the first RU486 induction; mIL12 protein level was measured by ELISA, and tumor volumes were measured using a calliper. At the end of the experiment we collected tibias, lungs and livers for immunohistochemical studies. Our results showed a reduction of local tumor growth and also a significant extended overall survival. Importantly, the treatment led to long-term survivors free of disease. We performed a re-challenge experiment to assess the generation of antitumor memory and we are currently waiting for those results. Immunohistochemical analyses showed an increase in CD3, mainly CD4 and CD8 and a decrease of T-reg cells. Moreover, splenocytes from treated mice produced significant higher amounts of IFNγ than those of control mice. In summary, the role of the microenvironment and the potent antitumor immune response mediated by controlled expression of IL12 by an HC-Ad equipped with a drug-inducible system can be a real therapeutic option for primary pediatrics OS. Citation Format: Marta Zalacain, Virginia Laspieda, Naiara Martinez-Vélez, Lucía Marrodán, María Buñuales, Marisol Gonzalez-Huarriz, Maider Varela, Montserrat Puigdelloses, Manuela Gonzalez-Aparicio, Marta María Alonso, Rubén Hernandez, Ana Patiño-García. High-capacity adenoviral vectors with controlled expression of interleukin 12 as a new strategy against pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3115.

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.

Age-related pathology after adenoviral overexpression of the leucine-rich repeat kinase 2 in the mouse striatum

Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration.

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.

Adaptation of vectors and drug-inducible systems for controlled expression of transgenes in the tumor microenvironment

Biological therapies based on recombinant proteins such as antibodies or cytokines are continuously improving the repertoire of treatments against cancer. However, safety and efficacy of this approach is often limited by inappropriate biodistribution and pharmacokinetics of the proteins when they are administered systemically. Local administration of gene therapy vectors encoding these proteins would be a feasible alternative if they could mediate long-term and controlled expression of the transgene after a single intratumoral administration. We describe a new vector platform specially designed for this purpose. Different combinations of transactivators and promoters were evaluated to obtain a fully humanized inducible system responsive to the well-characterized drug mifepristone. The optimal transactivator conformation was based on DNA binding domains from the chimeric protein ZFHD1 fused to the progesterone receptor ligand binding domain and the NFkb p65 activation domain. The expression of this hybrid transactivator under the control of the elongation factor 1α (EF1α) or the chimeric CAG promoters ensured functionality of the system in a variety of cancer types. Expression cassettes with luciferase as a reporter gene were incorporated into High-Capacity adenoviral vectors (HC-Ad) for in vivo evaluation. Systemic administration of the vectors into C57BL/6 mice revealed that the vector based on the EF1α promoter (HCA-EF-ZP) allows tight control of transgene expression and remains stable for at least two months, whereas the CAG promoter suffers a progressive inactivation. Using an orthotopic pancreatic cancer model in syngeneic C57BL/6 mice we show that the local administration of HCA-EF-ZP achieves better tumor/liver ratio of luciferase production than the intravenous route. However, regional spread of the vector led to substantial transgene expression in peritoneal organs. We reduced this leakage through genetic modification of the vector capsid to display RGD and poly-lysine motifs in the fiber knob. Safety and antitumor effect of this gene therapy platform was demonstrated using interleukin-12 as a therapeutic gene. In conclusion, we have developed a new tool that allows local, sustained and controlled production of therapeutic proteins in tumors.

18. Delivery of CRISPR/Cas9 or TALENs Against Hepatitis B Virus cccDNA by High-Capacity Adenoviral Vectors

High-capacity adenoviral vectors (HCAdV) are superior tools to deliver large DNA cargos into living cells. These vectors are devoid of all adenoviral coding sequences, leaving only essential adenoviral DNA sequences which are required for virus replication and packaging of HCAdV genomes containing foreign DNA. Here HCAdVs were utilized in combination with designer nucleases to develop a cure for chronic hepatitis B virus (HBV) infection. Although advanced prevention measures are available, chronic HBV infection still presents a serious global health burden for which no cure exists. The hepatitis B virus genome forms a persistent DNA species in infected cells (covalently closed circular DNA, cccDNA) and in that way is able to convert the infection into a chronic state. A chronic carrier state makes the sufferers susceptible for cirrhosis and liver cancer. To date various versions of designer nucleases against the HBV genome were already devised but yet no adequate transfer to HBV-infected cells in vitro and especially in vivo was presented. In the present study the potential of designer nucleases as a tool to cure chronic HBV infection was investigated and the aim was to cut and therefore destroy the HBV-DNA intracellularly. We employed transcription factor-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats / Cas9 (CRISPR/Cas9) system and additionally adapted HCAdV as an efficient delivery method. This is the advancement of this study in which either both TALEN subunits of a pair or multiple guide RNA expression cassettes alongside with a Cas9 coding sequence including promoter and termination signal were co-delivered in one vector. The expression cassettes including all compounds of both systems were inserted into HCAdVs. Vectors were amplified in large scale, their integrity monitored and then tested on liver cell lines which were infected with HBV. For this purpose we established an HBV infection model which is based on a HBV genome which was also inserted into a HCAdV. The effect of the designer nucleases on the HBV genome and its transcription was assayed by qPCR, a mutation detection assay and HBsAg ELISA. Our data revealed 80% reduction of hepatitis B surface antigen production in designer nuclease treated cells in comparison to untreated or mock treated cells. Furthermore cells treated with nucleases resulted in a decreased HBV genome copy number and the introduction of mutations could be demonstrated by a mutation detection assay using T7 endonuclease I. Note that the CRIRSPR/Cas9 system was superior to the TALEN based construct. In conclusion, we demonstrated delivery of a complete TALEN pair as well as a CRISPR/Cas9 construct containing three guide RNAs by use of just one HCAdV, respectively, which after application resulted in effective reduction of HBV parameters. Future objectives are to test our vectors in animal models of HBV infection and eventually to optimize the vector for the needs of this application.

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.

103. High-Capacity Adenoviral Vectors (HCAdV) Armed with a High Risk Human Papillomavirus (HPV) Oncogene Specific CRISPR/Cas9 Machinery Specifically Kill Cervical Cancer Cells

Persistent high risk human Papillomavirus (HPV) infections are the main cause of cervical cancer and partially also head and neck cancer. Recent studies showed that plasmid transfection of HPV oncogene specific designer nucleases effectively induced in/del mutations in the early promotors or oncogenes of HPV genomes integrated into the genome of cervical cancer cells by double-strand DNA break mediated non homologous end joining (NHEJ). Destruction or suppression of HPV oncogenes lead to decreased suppression of HPV oncogenes mediated by tumor suppressor protein p53 and retinoblastoma protein pRB inducing recovery from HPV oncogene mediated inhibition of apoptosis induction and cell cycle control. However, only few attempts have been made to improve delivery of respective designer nucleases by means of viral delivery allowing for translating these promising findings towards in vivo applications. Here we aimed at arming gene deleted high-capacity adenoviral vectors (HCAdVs) with HPV-specific CRISPR/cas9 machineries. By using a new toolbox that facilitates customization, cloning and production of CRISPR-HCAdVs we assembled HCAdV genomes containing the Streptococcus pyogenes Cas9 (spCas9) gene including either one gRNA expression unit specific for HPV18-E6 or two gRNA expression units specific for HPV18 and HPV16-E6 that have been shown efficiency to destroy respective genes. HPV specific CRISPR-HCAdVs were amplified in medium scale using a shortened protocol yielding high virus titers. Hela and Caski cervical cancer cells containing HPV18 or HPV16 genomes integrated into their cellular genome, as well as HPV negative A549 cells were infected with HPV specific CRISPR-HCAdVs. Adenoviral delivery of HPV specific CRISPR/Cas9 resulted in strong cell death in HPV positive cervical cancer cell lines whereas HPV negative A549 cells were unaffected. Moreover, HPV-specific CRISPR-HCAdVs infected Hela and Caski cells showed decreased proliferation compared to untreated cells and HPV negative control cells and cytotoxicity assays revealed strongly decreased cell viability of cervical cancer cells. In sharp contrast to control groups, increased apoptosis in HPV specific CRISPR-HCAdV treated cervical cancer cells was measured after performing apoptosis detection assays. Our results suggest that also HCAdVs can serve as oncolytic agents when armed with target specific designer nucleases such as CRISPR/Cas9. We believe that our approach will pave the way towards in vivo applications of CRISPR/Cas9 mediated oncolysis of HPV induced cervical cancer. As our CRISPR/Cas9-HCAdV production pipeline is adaptable to incorporate other or even more gRNA expression units specific for further HPV types, it provides a valuable platform to develop personalized antiviral or oncolytic vectors for any specific HPV target.