Krishna J. Fisher

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 β-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.

Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV

Recombinant adeno-associated virus (rAAV) is a promising vector for retinal application as it transduces photoreceptors and retinal pigment epithelium cells efficiently and in a stable fashion. Because rAAV also transduces retinal ganglion cells, we reasoned that ocular application of rAAV might result in delivery of transgenic protein to the CNS. Here we describe high levels of green fluorescent protein (GFP) persisting at least 6 months in optic nerves and brains of mice and dogs after intravitreal delivery of rAAV-GFP. There was no clinical or histological evidence of inflammatory response although a mild humoral Th-2 response to viral capsid proteins was detected. These findings have important implications with respect to therapeutic applications of rAAV.

Structural and functional heterogeneity of integrated recombinant AAV genomes

Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.

Titration of non-replicating adenovirus as a vector for transducing active TGF-β1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta1 (TGF‐β1) through a replication‐deficient recombinant adenovirus vector instilled into the lungs ( Sime et al. 1997 ). We have shown that this vector induces IPF in fibrogenic‐resistant tumour necrosis factor alpha‐receptor knockout (TNF‐αRKO) mice ( Liu et al. 2001 ). The object of our studies is to understand how peptide growth factors, such as TGF‐β1, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF‐β1 (AVTGFβ1) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF‐β1. The findings here show that 106 plaque‐forming units (pfu) of AVTGFβ1, provide essentially a ‘no‐effect’ dose, but even this amount of TGF‐β1 causes a significant increase in whole‐lung collagen by day 28 after treatment. In contrast, 108 and 109 pfu cause severe IPF in 4 days, whereas 107 and 5 × 107 are intermediate for all parameters studied, i.e. TGF‐β protein, inflammatory cells, cell proliferation, pro‐α 1(I) collagen gene expression and whole‐lung collagen accumulation, and expression of growth factors such as TGF‐β1, TNF‐α and PDGF‐A and ‐B. Interestingly enough, TGF‐β1, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF‐β1 biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.

A Preliminary Evaluation of Recombinant Adeno-Associated Virus Biodistribution in Rhesus Monkeys After Intrahepatic Inoculation In Utero

The ability to deliver genes to fetuses in utero may prove crucial for those genetic diseases that are associated with severe fetal morbidity and for which there is no effective postnatal therapy. In utero therapy may be especially useful in diseases that affect the central nervous system because the immature blood-brain barrier may facilitate gene delivery to neural target cells. We investigated whether in utero inoculation of recombinant adeno-associated virus (rAAV) into rhesus monkey fetuses would be a useful method of gene delivery, especially to the central nervous system. When the monkeys were sacrificed after birth, we found vector genomes distributed in many tissues, including the brain and peripheral blood. Pericapillary astrocytes expressing transgene products were detected by immunohistochemistry. In addition, we occasionally found vector genomes in the maternal blood. No adverse clinical or pathologic effects were observed in the inoculated monkeys. We concluded that (1) in utero intrahepatic inoculation of rAAV is a potentially safe and useful method of delivering genes to many fetal tissues; (2) astrocytes may be the cell type most easily targeted in the central nervous system (CNS) after systemic administration; and (3) the potential of inadvertent gene transfer to the mother must be considered.

Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects.