Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-03-07
2001-05-08
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S456000, C435S457000, C435S320100
Reexamination Certificate
active
06228646
ABSTRACT:
INTRODUCTION
BACKGROUND
Adenoviruses (Ads) belong to the family Adenoviridae and the human Ads belong to the genera Mastadenovirus. Human Ad infections are found worldwide. Ads were initially characterized in 1953 by Rowe et al. when trying to cultivate epithelial cells from the adenoids. The 47 different serotypes are grouped (A-F) according to their ability to cause tumours in newborn hamsters. Respiratory epithelial cells are the primary target for Ads in vivo. 5% of the acute respiratory diseases in children under the age of 5 are due to Ads. Other sites of infection include the eye, the gastro-intestinal tract and the urinary tract. Many Ad infections are subclinical and only result in antibody formation.
Three loosely defined sets of protein exist in the mature Ad: proteins that form the outer coat of the capsid, scaffolding proteins that hold the capsid together and DNA-binding proteins. The diameter of the icosahedral-shaped capsid varies from 65 to 80 nm depending on the serotype. The capsid is composed of a total of 720 hexon and 60 penton subunit proteins, 360 monomers of polypeptide VI, 240 monomers of polypeptide IX, and 60 trimeric fibre proteins.
Bound to the penton subunits and protruding from the capsid is the fibre protein which mediates the initial attachment of the virus to a target cell. Polypeptides IX, IIIa, and VI form the scaffolding which holds the capsid together. Polypeptide IX stabilizes the packing of adjacent hexons in the capsid, polypeptide IIIa spans the capsid to link hexons of adjacent faces, and polypeptide VI connects the structural proteins to the core. The core consists of DNA associated with polypeptides V, VII, &mgr; and the terminal protein.
Ads contain double stranded DNA as their genetic material. The base composition of the 47 characterized serotypes (Ad1-Ad47) varies in the percent G+C content and in the length of the genome (approximately 36 kb) and of the inverted terminal repeats (100-140 bp). The genome is covalently linked at each 5′ end to individual 55 kd terminal proteins, which associate with each other to circularize the DNA upon lysis of the virion.
The Ad genome is functionally divided into 2 major non-contiguous overlapping regions, early and late, based on the time of transcription after infection. The early regions are defined as those that are transcribed before the onset of viral DNA synthesis. The switch from early to late gene expression takes place about 7 hours after infection. The terms early and late are not to be taken too literally as some early regions are still transcribed after DNA synthesis has begun.
There are 6 distinct early regions; E1a, E1b, E2a, E2b, E3, and E4, each (except for the E2a-b region) with individual promoters, and one late region, which is under the control of the major late promoter, with 5 well characterized coding units (L1-L5). There are also other minor intermediate and/or late transcriptional regions that are less well characterized, including the region encoding the viral-associated (VA) RNAs. Each early and late region appears to contain a cassette of genes coding for polypeptides with related functions. Each region is transcribed initially as a single RNA which is then spliced into the mature mRNAs. More than 30 different mature RNA transcripts have been identified in Ad2, one of the most studied serotypes.
Once the viral DNA is inside the nucleus, transcription is initiated from the viral E1a promoter. This is the only viral region that must be transcribed without the aid of viral-encoded trans-activators. There are other regions that are also transcribed immediately after cell infection but to a lesser extent, suggesting that the E1 region is not the only region capable of being transcribed without viral-encoded transcription factors. The E1a region codes for more than six polypeptides. One of the polypeptides from this region, a 51 kd protein, transactivates transcription of the other early regions and amplifies viral gene expression. The E1b region codes for three polypeptides. The large E1b protein (55 kd), in association with the E4 34 kd protein, forms a nuclear complex and quickly halts cellular protein synthesis during lytic infections. This 55 kd polypeptide also interacts with p53 and directly inhibits its function. A 19 kd trans-activating protein encoded by the E1B region is essential to transform primary cultures. The oncogenicity of Ads in new-born rodents requires the E1 region. Similarly, when the E1 region is transfected into primary cell cultures, cell transformation occurs. Only the E1a region gene product is needed to immortalize cell cultures.
The E2a and E2b regions code for proteins directly involved in replication, i.e., the viral DNA polymerase, the pre-terminal protein and DNA binding proteins. In the E3 region, the 9 predicted proteins are not required for Ad replication in cultured cells. Of the 6 identified proteins, 4 partially characterized ones are involved in counteracting the immune system; a 19 kd glycoprotein, gp19k, prevents cytolysis by cytotoxic T lymphocytes (CTL); and a 14.7 kd and a 10.4 kd/14.5 kd complex prevent, by different methods, E1a induced tumour necrosis factor cytolysis. The E4 region appears to contain a cassette of genes whose products act to shutdown endogenous host gene expression and upregulate transcription from the E2 and late regions. Once viral DNA synthesis begins, the late genes, coding mainly for proteins involved in the structure and assembly of the virus particle, are expressed.
Recombinant human adenoviruses have attracted much attention of late because of their potential for gene therapy and gene transfer and for protein expression in mammalian cells. First-generation recombinant adenovirus vectors most often contain deletions in the E1a and/or E1b regions. The usefulness of such vectors for gene transfer has been demonstrated in mice, cotton rats and nonhuman primates (Engelhardt et al. Hum. Gene Ther. 4:759-769 1993; Rosenfeld et al. Cell 68:143-155 1992; Yang et al. Nat. Genet. 7:362-369 1994). A fundamental problem encountered in using these vectors for gene therapy, however, is that deletion of the E1 sequences alone is not sufficient to completely ablate expression of other early and late viral genes or to prevent replication of the viral DNA. Studies have indicated that these vectors express viral antigens which elicit destructive immune responses in the target cells (Yang et al. Proc. Natl Acad. Sci. 91:4407-4411 1994; Yang et al. Nat. Genet. 7:362-369; Yang et al. J. Virol. 69:2004-2015 1995). This immune response leads to loss of transgene expression and development of inflammation. In addition, there is indication that memory-type immune responses may substantially diminish the efficiency of gene transfer following a second and subsequent administrations of the recombinant vector (Kozarsky et al. J. Biol. Chem. 269:1-8 1994; Smith et al. Nat. Genet. 5:397-402 1993). Newer recombinant adenovirus vectors contain additional disabling mutations in other regions of the adenovirus genome, for example in E2a (Englehardt et al. Hum. Gene Ther. 5:1217-1229 1994; Englehardt et al. Proc. Natl Acad. Sci. 91:6196-6200) or E3 (Bett et al. Proc. Natl Acad. Sci. 91:8802-8806 1994). These vectors, although they express fewer viral proteins, do not completely eliminate adenoviral protein expression and so are subject to similar immune response problems as found with the earlier vectors.
In addition to the immune response problems associated with the use of the current adenovirus-based gene therapy vectors, only relatively small amounts of foreign DNA (that is, non-adenovirus DNA) can be accommodated in these vectors due to the size constraints of adenoviral packaging. Studies have shown that adenovirus virions can package up to approximately 105% of the wild type adenovirus genome length (the wild type adenovirus genome is between 35-36 kilobases). Recombinant vectors having deletions in the E1 region typically permit the insertion of less than 5 kb of foreign DNA. Recombinant vectors having additiona
Crouch Deborah
Mandel and Adriano
The Regents of the University of California
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