Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.

Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (delta vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases.

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.

COUP-TF plays a dual role in the regulation of the ovalbumin gene.

The ovalbumin (Ov) gene contains a number of regulatory elements that control its transcriptional activity and restrict expression to avian oviduct. One major regulatory region, the steroid-dependent regulatory element (SDRE), is required for induction by estrogen and corticosterone. Another region, the negative regulatory element (NRE), downstream of the SDRE, acts primarily to repress gene expression. In addition, experiments within indicate that the binding site for the COUP transcription factor (COUP-TF) is also required for Ov gene transcription. To examine the interactions involving the SDRE, the NRE, and the COUP binding sites on Ov gene transcription, mutations in these regions were made and transfected into primary oviduct cell cultures. These experiments show that without the NRE, the SDRE is sufficient for induction by estrogen and corticosterone, irrespective of the COUP site. However, with the NRE intact, the COUP site is required for steroid induction, although without the NRE, the COUP site attenuates transcriptional activity. More interestingly, overexpression of COUP-TF1 with the Ov wild-type reporter construct alleviates the requirement for steroid hormones. These results demonstrate that the COUP site is essential and has a dual role in Ov gene transcription and that steroid hormones might directly or indirectly regulate the activity of COUP-TF1.

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.