Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice.

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.

Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.

Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity.

The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas. In these cancers, the viral DNAs are often integrated into the host genome so that expression of the E1 and the E2 genes is lost, suggesting that disruption of these regulatory genes plays an important role in carcinogenic progression. Previous studies defining the viral genes affecting HPV-16 transformation functions have used the "prototype" viral genome, which was cloned from a human cervical carcinoma and later discovered to harbor a mutation in the E1 gene. In this study, we have corrected this mutation and have evaluated the effect of mutations of either the E1 or the E2 gene on the efficiency of HPV-16 immortalization of human keratinocytes. Mutation of either the E1 gene or the E2 gene in the background of a "wild-type" HPV-16 genome markedly increased immortalization capacity. Mutations were also generated in the E2-binding sites located upstream of the P97 promoter, which directs synthesis of the viral E6 and E7 transforming genes. E2 negatively regulates the P97 promoter through binding at adjacent sites. Surprisingly, the mutation of these sites only partially relieved the negative effect of E2 on viral immortalization, implicating additional mechanisms in the E2 repression of viral immortalization functions. Our results provide genetic evidence that the E1 and E2 gene products each can repress HPV-16 immortalization and support the hypothesis that a selective growth advantage is provided by integration of the viral genome in a manner that causes the loss of expression of either E1 or E2.

The viral transcriptional regulatory region upstream of the E6 and E7 genes is a major determinant of the differential immortalization activities of human papillomavirus types 16 and 18.

The human papillomavirus types 16 (HPV-16) and 18 (HPV-18) can immortalize primary human keratinocytes. The region of the viral genome responsible for this function maps to the E6 and E7 genes and their respective upstream transcriptional regulatory sequences, the long control regions (LCRs). The HPV-18 LCR/E6/E7 is more efficient in this immortalization function than the analogous region of the HPV-16 genome, resembling the difference in the immortalization potentials of the two full-length viral genomes. This study was designed to examine the basis for the difference in HPV-16 and HPV-18 immortalization efficiencies. The E6 and E7 genes of either HPV-16 or HPV-18, when expressed from the same heterologous promoter, immortalized primary human keratinocytes with the same low efficiency, suggesting that the difference in immortalization activities was not due to the different E6 or E7 genes themselves but rather to a difference in the transcriptional regulatory regions upstream of these genes. The analysis of a series of chimeric HPV-16 and HPV-18 LCR/E6/E7 constructs confirmed this observation and further mapped the viral element responsible for the major difference in immortalization efficiency to the transcriptional regulatory region upstream of the E6 and E7 genes.

Cellular targets of the oncoproteins encoded by the cancer associated human papillomaviruses.

Insight into the mechanisms by which DNA tumor viruses transform cells has come from the recognition that the virus-encoded oncoproteins interact specifically with important cell regulatory proteins. The "high risk" human papillomaviruses such as HPV-16 and HPV-18 which are associated with human anogenital carcinomas encode two transforming genes (E6 and E7) which are expressed in HPV positive cancers and derived cell lines. E7 shares functional and structural features with the adenovirus E1A proteins. Like Ad E1A and the large T proteins of the polyomaviruses, E7 can complex pRB. The E7 proteins of the "high risk" HPVs associate with pRB with approximately a 10-fold higher affinity than do the E7 proteins of the "low risk" HPVs, and important biological differences between the E7 proteins of these two groups of HPVs are determined by amino-terminal sequences which include the pRB binding domain. Like SV40 large T and Ad 5 E1B, the E6 oncoprotein encoded by the "high risk" HPVs can form a complex with p53. In vitro, E6 promotes the degradation of p53 and this degradation involves the ubiquitin-dependent protease system. The selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant acting oncoproteins. The relevance of the inactivation of the normal functions of pRB and p53 in human cervical carcinogenesis has recently been demonstrated by the analysis of these two genes and their products in a series of HPV-positive and HPV-negative cell lines. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild type p53 proteins, which are presumed to be altered in function as a consequence of association with the HPV oncoproteins. In contrast, mutations were identified in the p53 and RB genes expressed in the HPV-negative cervical carcinoma cell lines, C33-A and HT-3. These results support the hypothesis that the inactivation of the normal functions of the tumor suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or through complex formation with HPV E6 and E7 oncoproteins.

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

Association of amplified oncogene c-myc with an abnormally banded chromosome 8 in a human leukaemia cell line.

Several unusual chromosome structures have been described in drug-resistant cell lines and in certain tumours. These structures include elongated homogeneously staining regions (HSRs), small extrachromosomal paired chromatin bodies (double minutes, DMs) and abnormally banded regions (ABRs) with strong but anomalous band patterns. There is evidence that these are alternative forms of gene amplification, with HSRs breaking down to form DMs, and DMs integrating into the chromosome to generate HSRs and ABRs. Recently, it was demonstrated that, compared with several normal and leukaemia human cells, DNA sequences representing the human homologue of the onc gene of the avian myelocytomatosis virus (MC29), the so-called c-myc gene, were amplified in HL-60 cells. This is a human promyelocytic leukaemia cell line established in the laboratory of one of us (R.C.G.) at the National Cancer Institute (Bethesda, Maryland) in 1977, and widely used for studies on myeloid and monocytic differentiation. Amplification of the gene was present in primary leukaemic cells of the patient, and DMs were noted in some of these cells as well as in early passages of the HL-60 line. No structure resembling HSRs or ABRs were noted in karyotypic studies at this early stage and there were no alterations involving the long arm of chromosome 8 (8q), to which the c-myc gene has recently been mapped. We have now re-examined the karyotype of the HL-60 line, using cells frozen at various times during its continuous passage at the Wistar Institute (Philadelphia, Pennsylvania) to look for chromosomal abnormalities that might be associated with the amplification of c-myc. We find that, beginning in 1979, HL-60 cells at the Wistar Institute no longer had DMs, but did show an abnormal 8q+ chromosome, replacing a normal chromosome 8, and representing an ABR reflecting the site of myc gene amplification.