-
[show abstract]
[hide abstract]
ABSTRACT: Helper-dependent adenoviral vectors (HDAd) can mediate long-term phenotypic correction in the ornithine transacarbamylase (OTC)-deficient mice model with negligible chronic toxicity. However, the high doses required for metabolic correction will result in systemic inflammatory response syndrome in humans. This acute toxicity represents the major obstacle for clinical applications of HDAd vectors for the treatment of OTC deficiency. Strategies for reducing the dose necessary for disease correction are highly desirable because HDAd acute toxicity is clearly dose-dependent.
We analysed a potent expression cassette and the hydrodynamic injection for the ability to reduce the HDAd dose necessary for phenotypic correction in OTC-deficient spf-ash mice.
We have developed a vector containing a potent expression cassette expressing the OTC transgene, which allowed phenotypic correction at lower doses. Our results suggest that vector expressing greater OTC levels allows correction of orotic acid overproduction at lower doses that make clinical translation more relevant. We were able to further reduce the minimal effective dose by delivering the vector through the hydrodynamic injection technique.
Vectors containing the expression cassette used in the present study, combined with other strategies for improving HDAd therapeutic index, will likely permit application of these vectors for the treatment of OTC deficiency as well as other urea cycle disorders.
The Journal of Gene Medicine 06/2008; 10(8):890-6. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bioengineering of the factor VIII (FVIII) molecule has led to the production of variants that overcome poor secretion and/or rapid inactivation. We tested six modified FVIII variants for in vivo efficacy by expressing them from helper-dependent adenoviral (HD-Ad) vectors. We constructed a wild-type (WT) variant, a B-domain-deleted (BDD) variant, a point mutant for improved secretion (F309S), a variant with a partial B-domain deletion for improved secretion (N6), a combination of the point mutant and partial BDD variant (F309N6), and an inactivation-resistant (IR8) FVIII variant. All the constructs expressed functional protein after injection of high-dose HD-Ad. Activity ranged from 20 to 50% with WT, to approximately 100% with the N6 and F309N6 variants. Interestingly, mice treated with N6 showed long-term FVIII activity and phenotypic correction for up to 74 weeks, with low anti-FVIII antibody titer. Importantly, the N6 variant was therapeutically efficacious even after a 50% reduction of viral dose, thereby indicating that transgene modification itself can improve the dose efficacy of HD-Ad. This finding is significant, because dose efficacy is a key factor in clinical application. In summary, bioengineering of the FVIII molecule may be an effective approach to improving the safety, immunogenicity, and efficacy of HD-Ad gene therapy in hemophilia A (HA).
Molecular Therapy 01/2008; 15(12):2080-7. · 6.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Dendritic cells (DCs) are essential for initiating and directing antigen-specific T-cell responses. Genetic modification of DC is under study for cancer immunotherapy, vaccine development, and antigen-targeted immunosuppression. Adenovirus (Ad) type 5 (Ad5)-mediated gene transfer to mouse bone marrow DCs and human monocyte-derived DCs is inefficient because neither express the cognate high-affinity Ads receptor. We show that co-precipitating adenoviral vectors with calcium phosphate (CaPi) increased gene expression (2000-fold) and transduction efficiency (50-fold) in mouse DC, primarily owing to receptor-independent viral uptake. Moreover, Ad5:CaPi-treated DCs were activated to express the maturation surface molecules CD40 and CD86, and to secrete proinflammatory cytokines tumor necrosis factor-alpha and interleukin 6. However, neither DC transduction nor maturation was dependent on viral protein interactions with cell surface integrin. Ad5:CaPi also transduced human DC more efficiently than Ad5 alone, similar to a genetically modified vector (Ad5f35) targeted to the CD46 receptor. As such, this approach combines the efficiency of adenoviral-mediated endosomal escape and nuclear trafficking with the receptor independence of nonviral gene delivery. Importantly, CaPi co-precipitation could be used to functionally modify DC to activate and expand cytomegalovirus-specific memory cytotoxic T lymphocytes. This study identifies a simple technique to improve the efficacy of current Ad5 gene transfer, in support of clinical adoptive immunotherapy.
Molecular Therapy 03/2007; 15(2):386-92. · 6.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.
Molecular Therapy 03/2007; 15(2):378-85. · 6.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Molecular Therapy (2005) 11, S231|[ndash]|S231; doi: 10.1016/j.ymthe.2005.07.138
598. In-Vivo Test of Coagulation Factor VIII with Improved Properties for Hemophilia A Gene Therapy by Helper-Dependent Adenoviral Vectors
Vincenzo Cerullo1, Michael McCormack1, Racel Garcia1, Christian Clarke1, Steven W. Pipe2 and Brendan Lee11Molecular and Human Genetics, Baylor College of Medicine, Houston, TX2Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI
Molecular Therapy 04/2005; · 6.87 Impact Factor
-
-
