Gene-deleted Adenoviral Vectors Research Articles

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery.

Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.

Exploring gene‐deleted adenoviral vectors for delivery of short hairpin RNAs and reduction of hepatitis B virus infection in mice

RNA interference based therapeutic approaches hold promise for the treatment of patients chronically infected with hepatitis B virus (HBV). To conquer HBV infection, long-term suppression of target transcripts in all hepatocytes without toxic effects may be required. The present study explored gene-deleted adenoviral vectors (GD-AdV) lacking all viral coding sequences for delivery of the previously described short hairpin RNA (shRNA) HBVU6no.2, which was demonstrated to result in post-transcriptional knock-down of HBV transcripts. We established conditions for shRNA delivery expressed from GD-AdV in vitro and in vivo and observed up to 96% shRNA-mediated knockdown of luciferase expressed in mouse liver. To investigate in vivo efficacy of HBVU6no.2 expressed from a GD-AdV, we explored a transient and a transgenic mouse model for HBV infection. We observed an up to 68% drop in serum HBV surface antigen (HBsAg) levels in the transient and the transgenic mouse model for HBV infection, respectively. Interestingly, we detected an up to 86% drop in HBsAg levels in both animal models after administration of a control GD-AdV encoding beta-galactosidase. In concordance with reduced serum HBsAg levels, we observed reduced HBV replication as demonstrated by Southern blot analysis of HBV genomes. The present study demonstrates that GD-AdV can be used against HBV infection but the design of DNA sequences including shRNAs contained in the vector and virus-host interactions during superinfection needs to be carefully considered.

9. Linear Monomers Are Responsible for Persistence of Gene-Deleted Adenoviral Vectors In Vitro and in Mouse Liver

9. Linear Monomers Are Responsible for Persistence of Gene-Deleted Adenoviral Vectors In Vitro and in Mouse Liver

Somatic Integration From an Adenoviral Hybrid Vector into a Hot Spot in Mouse Liver Results in Persistent Transgene Expression Levels In Vivo

We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.

852. Chromatin Study of Adenovirus Vector DNA In Vivo

Recombinant adenoviral vectors are robust at transgene expression in a wide variety of tissues including the liver. While adenoviral- mediated transgene expression can be persistent in the absence of toxicity, very little is know about the structure of the adenoviral genome once it enters the nucleus. Our interest is related to the chromatin structure of both 1st generation and gene-deleted adenoviral vectors in vivo. We have previously established that intact plasmid DNAs that are silenced have a different chromatin structure than minicircle DNAs devoid of bacterial plasmid DNA sequences, which are persistently active in vivo.

11. Transposition from a Gene-Deleted Adenoviral Vector Results in Phenotypic Correction in a Canine Model for Hemophilia B

Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences offer high transduction efficiencies but were hampered by transient phenotypic correction at a non-toxic dose. For persistent hemostatic correction in hemophilia B dogs, our current study utilized a novel gene-deleted adenoviral vector system which combines adenoviral vectors for high transduction efficiencies and for the first time in a larger animal the Sleeping Beauty (SB) transposase for somatic integration and stable transgene expression. We generated the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 in which a transposon with a canine factor IX (cFIX) expression cassette is flanked by FRT sites. In the presence of Flp recombinase and the recently developed hyperactive transposase HSB5 (Yant et al., unpublished), the transgene undergoes Flp mediated excision followed by HSB5 mediated somatic integration.To analyze transgene persistence in vivo C57Bl/6 mice were co-transfused with 2x10e9 transducing units (TU) of the vector FTC/TcFIX/attB/(FRT)2 and 7×10e8 TU of a second HD vector which encodes HSB5 and Flp. Control mice received an inactive version of SB, respectively. Rapid cell cycling of mouse hepatocytes was induced by performing a two-thirds partial hepatectomy and by injecting CCl(4). Seventy-five days post-injection we detected serum levels of cFIX of up to 2000 ng/ml in the active SB group and no cFIX in the control mice. This indicated that integration of the transgene from the episomal adenoviral vector genome into the host genome occurred.To test for persistence of hemostatic correction of the bleeding diathesis in hemophila B dogs we have co-injected 5.4x10e11 TU of the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 and 2.6x10e11 TU of the HSB5 and Flp encoding vector. This equals a total dose of 1.6×10e13 viral particles (vps) and 1x10e12 vps/kg body weight. We measured plasma cFIX levels of up to 3900ng/ml (normal level = 5000ng/ml) and observed complete phenotypic correction of the factor IX deficiency. The whole blot clotting time (WBCT) was reduced from 60min to 18.5min on day 35, the length of the study to date. This was in sharp contrast to our previous studies in hemophilia B dogs in which we used a non-integrating HD vector. In that study we observed a 3-fold increase of the WBCT 35 days post-injection which indicates that in our current study transposition stabilized phenotypic correction. In contrast to previous studies using early generation adenoviral vectors and keeping in mind that for the first time the in vivo performance of the SB transposase in a larger animal was evaluated, we observed no vector-related elevation of liver enzymes and no fall in platelet counts. Taken together, this study demonstrates that this adeno-transposon hybrid vector system will be important for treating genetic diseases.

441. Short Hairpin RNA Interference Expressed from a Gene-Deleted Adenoviral Vector Results in Reduction of Hepatits B Surface Antigen Levels In Vitro and in a Small Animal Model for Hepatitis B Infection

Top of pageAbstract There is substantial need for new treatment strategies for chronically hepatitis B virus (HBV) infected patients. This study explores the rational design of novel adenoviral gene therapy vectors for efficient delivery of RNA interference (RNAi) inhibitor molecules against HBV. Over the recent years various viral and non-viral vectors have been studied for delivery of short hairpin RNAi (shRNAi) expression cassettes but the in vitro and in vivo efficacy of significantly safer gene-deleted adenoviral vectors (HD vectors) for delivery of shRNAi remains to be tested. To show in vivo efficacy regular FVB mice (n=3 per group) were co-injected with a HD vector with a shRNAi expression cassette against luciferase and a second adenoviral vector expressing luciferase. We found up to 96% reduction of luciferase expression seven days post-injection. Our laboratory previously demonstrated that a 25-mer shRNAi against HBV (HBVU6no.2) driven by a polymerase III promoter was sufficient to inhibit HBV in a non-viral gene therapy approach in vivo (McCaffrey et al., Nat Biotechnol 21:639-644, 2003) but was lethal in the context of a novel self-complementery adeno-associated viral vector (AAV) in HBV transgenic and normal mice (Grimm et al., 2004). In this study we created a gene-deleted adenoviral vector with the same shRNAi expression cassette (FTC/HBVU6no.2). In cells stably transfected with the HBV genome we observed up to 60% reduction of secreted surface antigen (sAg) levels, a marker for progression of HBV infection. In a further step we transfused a plasmid containing the HBV-genome into mouse liver of C57Bl/6 mice (n=4 per group) by high pressure tail vein injection followed by injection of 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2. This viral dose was sufficient to transduce 100% of all hepatocytes. We observed a 70% drop in serum sAg levels. Control mice which received only the HBV containing plasmid or a HD vector with a beta-galactosidase (|[beta]|-Gal) expression cassette showed a 29% and a 73% drop in serum sAg levels, respectively. HBV transgenic mice (n=4 per group) were either injected with 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2 or a |[beta]|-Gal expressing HD vector. After a transient increase in sAg levels in both groups, we observed an up to 45% and 73% reduction in serum sAg levels compared to the vehicle control group, respectively. Limited toxicity studies were performed and highest liver enzyme levels were measured 20 days post-injection which returned to normal levels 40 days post-injection for the FTC/HBVU6no.2 but not the |[beta]|-Gal group. We speculate that an immune response against the transgene |[beta]|-Gal is responsible for the significant drop in serum sAg levels and the sustained increased liver enzyme levels. In summary, this study demonstrates that gene-deleted adenoviral vectors can be used to deliver small inhibitor molecules in vivo and with further improvements these vectors will play an important role in gene therapy approaches based on shRNAi.

600. A Novel Gene-Deleted Adenoviral Vector for Somatic Integration and Long-Term Phenotypic Correction of Murine and Canine Hemophilia B

This study describes the development of a novel gene-deleted adenoviral vector system for somatic integration. This system utilizes either a very recently generated hyperactive Sleeping Beauty transposase (HSB5) or a bacteriophage integrase derived from Streptomyces (phiC31). Previous data from our laboratory revealed that integration efficiencies for both recombinases are higher from circular substrates compared to linear substrates. Thus, we generated the gene-deleted adenoviral vector FTC/TcFIX/attB-(FRT)2 in which a canine factor IX (cFIX) expression cassette with the phiC31 recognition site attB and the transposase inverted repeats (IR) for HSB5 mediated integration are flanked by FRT sites for Flp mediated circle formation from the recombinant adenoviral vector.

821. Development of a Site-Specific Integrating Gene-Deleted Adenoviral Vector for Sustained Transgene Expression Levels In Vivo

Integrating vectors have been demonstrated to result in sustained transgene expression levels in vivo, but most are hampered by the risk of insertional mutagenesis. For a safer gene therapy protocol, site-specific integrating vectors are needed. Recently, it was shown that integration mediated by the integrase phiC31 derived from a Streptomyces phage resulted in site-specific integration by recombining the phiC31 recognition site attB in an episomal plasmid with pseudo attP sites in the host chromosome (Olivares et al., Nat Biotechnol 20:1124-8, 2002). In this study, we present a hybrid vector which utilizes the high transduction efficiency of a gene-deleted (HD) adenoviral vector to deliver the site-specific integration machinery of phiC31. Recombinant adenoviral vector genomes mainly persist as linear DNA molecules which motivated us to first compare the phiC31 mediated integration efficiency from circular and linear episomal DNA. We found that the integration efficiency was 14.8-fold higher from circular substrates and only 3.8-fold higher from linear DNA molecules compared to the mutant integrase control group. Thus, we based our system on a two vector system in which the transgene expression cassette is circularized from a HD vector by Flp mediated recombination, followed by phiC31- mediated integration.

A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia

AbstractMany approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity. (Blood. 2003;102:2403-2411)

Comparison of viral gene-deleted adenoviral vectors with the E1-deleted adenoviral vector in islets

Comparison of viral gene-deleted adenoviral vectors with the E1-deleted adenoviral vector in islets