Gene Therapy Of Chronic Disease Research Articles

546. Development of MFP-Inducible System for AAV5 Gene Therapy of Chronic Diseases in the Liver

Introduction: Gene therapy offers long term solutions for chronic diseases, whereby the transgene is continuously expressed upon single vector administration. However in some cases it would be desirable to tightly regulate or switch off transgene expression. Methods: We are investigating regulated gene expression based on the mifepristone (MFP)-inducible GeneSwitch system. The GeneSwitch protein comprises yeast Gal4 DNA-binding domain, a human p65 activation domain and a MFP controlled domain derived from the human progesterone receptor. The classical GeneSwitch system consists of two expression cassettes on two separate vectors; one containing the GeneSwitch sequence and one containing the transgene. We compared this two-vector system to a single-vector system, where the two cassettes were put into one vector for efficacy in vitro and in vivo. Results: We show inducible expression of EPO, IGF and GNDF obtained in vitro upon addition of MFP to cells transfected with plasmids containing GeneSwitch and the gene expression cassette. The kinetics of EPO mRNA and protein expression followed a dose dependent fashion in the range of 0.1 to 10 nM MFP and reached a plateau at higher MFP concentrations. Surprisingly, the GeneSwitch protein expression decreased 48h after MFP induction. The indicibility of the single versus the two-vector system of GeneSwitch-EPO was compared. Both systems were equally inducible based on total amount of EPO produced in the presence of MFP and related to background expression in the absence of MFP. In vivo proof of concept was obtained for EPO in the liver. EPO is characterized by clear expression kinetics in plasma and raises blood hematocrit, hence provides a reliable in-life read-out for gene inducibility. Mice were injected with different doses of AAV5-AAT-GeneSwitch-EPO and gene expression was induced in two separate rounds at 4 and 8 weeks p.i. EPO plasma levels increased approximately 2-logs in the single or two-vector system-injected mice, compared to un-induced groups. Moreover in the absence of MFP background expression of EPO was lower in the single-vector system and hematocrit levels were unaffected. Measurements of MFP in tissue matrices and in plasma by mass spectrometry show the presence of MFP in plasma and liver, validating applicability of the GeneSwitch system in the liver. Conclusion: Overall, our data indicate that transgene expression can be repeatedly regulated in the liver using the GeneSwitch system and provides us with a novel AAV5 vector for further development.

Biophysical and biological investigation of DNA nano-complexes with a non-toxic, biodegradable amine-modified hyperbranched polyester

Designed for gene therapy of chronic diseases, HBP-DEAPA 60 is a non-toxic biodegradable amine modified hyperbranched polyester. This candidate was chosen from a series of hyperbranched polymers for further characterization as it showed the best transfection efficiency and fastest degradation rate. HBP-DEAPA 60/DNA complexes were investigated with regard to stability, uptake and formation to gain a better insight into HBP-DEAPA 60/DNA complex properties. We investigated HBP-DEAPA 60/DNA complex uptake into A 549 cells by FACS and CLSM. Their stability was investigated by a heparin displacement assay as well as by DNAse I assay. Morphology was shown by AFM. HBP-DEAPA 60/DNA complex formation was further characterized in terms of thermodynamic parameters. We studied the conformation of DNA in nano-complexes via circular dichroism (CD) spectroscopy for different NP ratios. Thermodynamic studies showed that binding enthalpies were endothermic; the nano-complex formation was entropically driven. Although PEI/DNA and HBP-DEAPA 60/DNA complexes showed similar behavior with regard to uptake, heparin stability, DNA helicality and their entropically driven complex formation they differ in their binding constant K(a) and in their ability to protect the DNA from DNAse. Concerning K(a) and DNAse stability, HBP-DEAPA/DNA complexes should be further optimized. This shows that different characterization studies are necessary to fully characterize polyplex stability and properties.

Nonviral Gene Therapy Approaches to Hemophilia

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.

Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.

Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8(+) cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.

Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues

First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.