Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector.

Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in ß-glucuronidase (ß-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for ß-glu (GUSB) in the canine MPS VII cornea. ß-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of ß-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain.

Severe deficiency in lysosomal ß-glucuronidase (ß-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in ß-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, ß-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

Corneal transduction by intra-stromal injection of AAV vectors in vivo in the mouse and ex vivo in human explants.

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.

A real-time PCR assay for quantification of canine adenoviral vectors.

The pre-existing humoral and cellular immunity found in the great majority of the population raises concerns about the clinical efficacy and safety of vectors derived from ubiquitous human adenovirus serotypes. To alleviate these concerns, canine adenovirus type 2 vectors (CAV-2) were developed. Owing to their extraordinary neuronal tropism and efficient axonal retrograde transport, CAV-2 vectors hold great promise for the treatment of neurodegenerative diseases. The development and validation of a SYBR Green qPCR assay for determination of CAV-2 titers is reported in the present study. This method uses specific primers designed to amplify a small genomic fragment of CAV-2 structural genes (pVI-hexon). The method was accurate and reproducible as determined by the low intra- and inter-assay variability (<15% R.S.E.). It is sensitive and useful over a 5-log range (1 x 10(3) to 1 x 10(7)genome copies/reaction). The assay can be used to quantify purified vector samples as well as crude viral lysates. The titers obtained by qPCR correlated well with both, those obtained by OD(260) and TCID(50) as indicated by the high coefficients of determination obtained by regression analysis (r(2)>0.83). The development of this simple and rapid CAV-2 quantitation method should be helpful for process development and monitoring.

The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.

Gene transfer may be preventive but not curative for a lysosomal transport disorder.

Cystinosis belongs to a growing class of lysosomal storage disorders (LSDs) caused by defective transmembrane proteins. The causative CTNS gene encodes the lysosomal cystine transporter, cystinosin. Currently the aminothiol cysteamine is the only drug available for reducing cystine storage but this treatment has non-negligible side effects and administration constraints. In this study, for the first time, we report viral vector-mediated CTNS gene transfer and evaluate the feasibility of this strategy as a complementary treatment. Initially, we transduced human CTNS(-/-) fibroblast cell lines and primary murine Ctns(-/-) hepatocyte cultures in vitro and demonstrated that gene transfer can reduce cystine storage. Because of age-related increase in cystine levels, we transduced hepatocytes from young (/=5 months of age) mice. Our in vitro data suggested that the efficiency of correction was age-dependent. We tested these observations in vivo: short-term (1 week) and long-term (4 weeks) CTNS-transduction significantly reduced hepatic cystine levels in young, but not older, Ctns(-/-) mice. Our data provide the proof-of-concept that gene transfer is feasible for correcting defective lysosomal transport, but suggest that, in the case of cystinosis, it could be preventive but not curative in some tissues.