Christine L. Halbert

Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors

ABSTRACT Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations ofrep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.

Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes

ABSTRACT Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.

Sheep retrovirus structural protein induces lung tumours

Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1–2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.

Successful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Original ArticleSuccessful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

Expression of human α1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.We evaluated the potential of lung-directed gene therapy for α1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

Persistent, Therapeutically Relevant Levels of Human Granulocyte Colony-Stimulating Factor in Mice after Systemic Delivery of Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) vectors have been shown to preferentially transduce hepatocytes after systemic administration in adult mice and to provide long-term expression of introduced genes. One application of this technology would be for the production of granulocyte colony-stimulating factor (G-CSF), which increases mature neutrophil numbers in humans and in animals, and has therapeutic effects in disorders featuring chronic neutropenia, including cyclic, severe congenital, and idiopathic neutropenia, and glycogen storage disease type Ib. We have treated mice by tail vein injection of AAV vectors encoding human G-CSF, and have detected high G-CSF levels and marked elevation of neutrophil counts for at least 5 months. A therapeutically relevant amount of G-CSF production was obtained when the liver-specific mouse albumin promoter-enhancer was used to drive G-CSF expression. In mice receiving higher amounts of vector, plasma levels of human G-CSF gradually increased over 3 weeks to high concentrations, whereas for lower amounts human G-CSF remained at initial, low levels. The previously observed effect of gamma irradiation, to increase AAV transduction rates, was diminished when large amounts of vector were used. Absolute neutrophil counts increased 10- to 50-fold for the period of observation to levels that would be therapeutic in the treatment of cyclic neutropenia. In conclusion, gene therapy with AAV vectors synthesizing G-CSF shows promise for the treatment of disorders featuring neutropenia.

CHARACTERIZATION OF IMMORTALIZED RABBIT LACRIMAL GLAND EPITHELIAL CELLS

SummaryTo establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm2 plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 µM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.

Envelope Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Induce Similar Bronchioalveolar Tumors in Lungs of Mice

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) induces bronchioalveolar tumors in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse airway epithelial cells induces similar tumors, indicating that Env expression is sufficient for tissue-specific tumor formation. Enzootic nasal tumor virus (ENTV) is related to JSRV but induces tumors in the nasal epithelium of sheep and goats. Here we found that ENTV Env can also induce tumors in mice but, unexpectedly, with a phenotype identical to that of tumors induced by the JSRV Env, indicating that factors other than Env mediate the tissue specificity of tumor induction by ENTV.