The contribution of the E6 and E7 open reading frames of human papillomavirus type 6b (HPV6b) and HPV16 to immortalization of human keratinocytes was evaluated by using amphotropic recombinant retroviruses. The HPV16 E7 gene could immortalize primary human keratinocytes without the cooperation of the viral E6 gene; however, E6 was able to contribute significantly to the efficiency of the E7 immortalizing function. Infection of HFE cells with retroviruses carrying the 16E6, 6bE6, 6bE7, or 6bE6E7 open reading frame did not result in immortalization.

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 of rep, 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.

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by asiation with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle checkpoint control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epitheial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decremd, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely-reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G, growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.

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.

Vectors based on recombinant adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV2) are the most thoroughly characterized and frequently employed. Here, we characterize transduction of the skeletal musculature using rAAV vectors pseudotyped with serotype 6 capsid proteins (rAAV6). We demonstrate that rAAV6 vectors can efficiently transduce the skeletal musculature of mice at levels >500-fold higher than is achievable with rAAV2 vectors and can readily saturate individual muscles following direct injection. Further, rAAV6 vectors are capable of transducing the diaphragm and intercostal muscles of mice after a simple injection into the intrathoracic cavity and are capable of widespread transduction throughout the musculature of mice injected in the intraperitoneal space as newborn pups. These results demonstrate that rAAV6 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.

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Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences 1 . 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 2 . Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses 3 , 4 , 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 adenoassociated 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.Oncogenic retroviruses are known to cause cancer by the acquisition and expression of hostderived oncogenes, by the insertional activation of host cell oncogenes or by the expression of auxiliary viral oncogenes such as the tax gene of human T-cell leukaemia virus. JSRV is a simple retrovirus (Fig. 1) that does not express a host-derived or auxiliary oncogene and can induce lung tumours in as little as 10 days 5 , a much shorter latency than typically found for the insertional activation of host oncogenes by other retroviruses. The mechanism of oncogenesis is unknown, but the JSRV Env protein has been found to transform cells in culture 2 , 6 -8 . One mechanism of transformation involves activation of the phosphoinositide-3-OH kinase (PI3K)/Akt pathway and is dependent on the presence of the cytoplasmic tail of Env 8 -10 , and the other involves Env binding to Hyal2, Hyal2 degradation, and activation of the RON receptor tyrosine kinase, which is normally suppressed by Hyal2 (ref. 11 ).Further studies of Env oncogenesis in animals are limited by the difficulty and expense of experimentation with a contagious oncogenic virus in sheep and by the inability of JSRV to infect convenient rodent animal models such as mice. However, we have found that adenoassociated virus (AAV) vectors made with AAV type 6 capsid proteins (AAV6 vectors) can promote long-term gene expression in all epithelial cell types in mouse lung 12 . To test whether Env alone would induce lung tumours we administered a mixture of 5 × 10 10 vector genomes of an AAV6 vector that expressed Env (ARJenv; Fig. 1) and 5 × 10 9 vector genomes of an AAV6 vector that expressed human placental alkaline phosphatase (AP) (ARAP4; Fig. 1 Fig. 2e, f) and a few tumours stained uniformly for AP (not shown), showing that occasional tumour progenitor cells were transduced by ...

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.