A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

DAP kinase is a pro-apoptotic calcium-regulated serine/threonine kinase, whose expression is frequently lost in human tumours. Here we show that DAP kinase counteracts oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint. Ectopic expression of DAP kinase suppressed oncogenic transformation of primary embryonic fibroblasts by activating p53 in a p19ARF-dependent manner. Consequently, the fibroblasts underwent apoptosis, characterized by caspase activation and DNA fragmentation. In response to c-Myc or E2F-1, the endogenous DAP kinase protein was upregulated. Furthermore, functional or genetic inactivation of the endogenous DAP kinase reduced the extent of induction of p19ARF/p53 and weakened the subsequent apoptotic responses to c-Myc or E2F-1. These results establish a role for DAP kinase in an early apoptotic checkpoint designed to eliminate pre-malignant cells during cancer development.

Recombinant adenoviruses (Ads) are highly efficient at transferring foreign genes to the liver in vivo; however, the duration of gene expression is limited by the host antiviral immune response, which prevents expression upon readministration of the virus. To test whether overexpression of the immunomodulatory products of the early Ad genome region 3 (E3) could prevent the antiviral immune response and prolong expression of foreign genes delivered by Ad vectors, we injected a recombinant Ad (Ad-E3-hBUGT), containing both E3 and the human bilirubin-uridine-diphosphoglucuronate-glucuronosyltransferase (BUGT) genes, into BUGT-deficient hyperbilirubinemic Gunn rats. Control Gunn rats received Ad-hBUGT, which expresses human BUGT alone. An initial injection of either virus resulted in hepatic expression of human BUGT as evidenced by excretion of bilirubin glucuronides in bile and a reduction of mean serum bilirubin levels from 7.0 mg͞dl to 1.9-2.7 mg͞dl within 7 days. In Ad-E3-hBUGT-injected rats, serum bilirubin levels increased to 4.5 mg͞dl by 84 days after infection, but a second administration of the virus on that day resulted in a hypobilirubinemic response similar to that seen with the first injection. In contrast, rats receiving Ad-hBUGT had serum bilirubin levels of 7 mg͞dl on day 84 after infection, but showed no reduction of serum bilirubin by reinjection of the virus on that day. In the rats injected with Ad-E3-hBUGT, but not in the ones injected with Ad-hBUGT, there was a marked inhibition of the antiviral antibody and Ad-specific cytotoxic T lymphocyte responses. This is the first demonstration that insertion of E3 genes in recombinant Ads facilitates readministration of a functional vector for long-term correction of an inherited metabolic disorder.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.genome engineering | conditional-null

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.

Recombinant adenoviruses (Ads) efficiently transfer foreign genes into hepatocytes in vivo, but the duration of transgene expression is limited by the host immune response which precludes gene expression upon readministration of the virus. To test if this immune response can be abrogated by oral tolerization, we instilled protein extracts of a recombinant adenovirus type-5 via gastroduodenostomy tubes into bilirubin-UDP-glucuronosyltransferase-1 (BUGT 1 )-deficient jaundiced Gunn rats. Control rats received BSA. Subsequent intravenous injection 5 ϫ 10 9 pfu of a recombinant adenovirusexpressing human BUGT 1 (Ad-hBUGT 1 ) resulted in hepatic expression of human BUGT 1 (hBUGT 1 ) with reduction of serum bilirubin levels by 70%. After 2 mo serum bilirubin increased gradually. In orally tolerized rats, but not in controls, a second dose of the virus on day 98 markedly reduced serum bilirubin again. In the tolerized rats, the development of antiadenoviral neutralizing antibodies and cytotoxic lymphocytes were markedly inhibited, and transplantation of their splenocytes into naive Gunn rats adoptively transferred the tolerance, indicating a role for regulatory cells. Lymphocytes from the tolerized rats hyperexpressed TGF ␤ 1 , IL2, and IL4 upon exposure to viral antigens, whereas IFN ␥ expression became undetectable. Thus, oral tolerization with adenoviral antigens permits long-term gene expression by repeated injections of recombinant adenoviruses. ( J. Clin. Invest. 1997. 99:1098-1106.)

In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.

Exposure to wild-type adenoviruses is common in humans and results in immune response against adenoviruses. The pre-existing antibodies and a strong secondary humoral and cellular immune response would interfere with gene transfer using recombinant adenoviral vectors. To test whether the secondary immune response can be abrogated by oral tolerization to adenoviral antigens, we immunized bilirubin-UDP-glucuronosyltransferase (BUGT)-deficient jaundiced Gunn rats with a recombinant adenovirus (5 ؋ 10 9 pfu/rat) expressing the human UDP-glucouronosyltransferase (BUGT 1 ) gene (Ad-hBUGT). Transgene expression was shown by reduction of mean serum bilirubin levels from 7.0 mg/dL to 2.3 mg/dL in 14 days, which then increased gradually to pretreatment levels in 6 weeks. All recipients developed antibodies (1:2 10 ) and cytotoxic lymphocytes against the adenovirus. For oral tolerization, we administered to the immunized rats protein extracts of a recombinant adenovirus type 5 (1-1.5 mg/day) via duodenostomy tubes 10 to 40 days after the initial virus injection; control rats received bovine serum albumin. In rats fed adenoviral proteins and the BSA-fed controls, the antibody titers decreased to 1:2 7 and 1:2 9 , respectively, in 70 days. Lymphocytes from the tolerized rats expressed TGF-␤ 1 upon exposure to antigen-presenting cells primed with adenoviral antigens, whereas IFN-␥ expression was undetectable. In contrast, lymphocytes from the BSA-treated control rats expressed IFN-␥ but not transforming growth factor ␤ 1 (TGF-␤ 1 ). Seventy days after the first injection in the orally tolerized rats, but not in the controls, a second Ad-hBUGT injection caused human BUGT 1 expression again, reducing serum bilirubin levels to those observed after the first injection. In the tolerized rats, serum antibody titers and anti-adenoviral cytotoxic lymphocyte activities continued to decline despite the second injection, whereas the antibody levels were boosted in the non-tolerized group. This results show that by preventing the secondary booster response, oral tolerization permits repeated adenovirusdirected gene transfer despite the presence of a residual antibody titer from a previous adenoviral exposure. (HEPA-TOLOGY 1998;27:1368-1376.)Recombinant adenoviruses are the most efficient vectors for gene transfer into quiescent hepatocytes in vivo. 1,2 We have shown that recombinant adenoviral vectors can transduce a sufficient number of hepatocytes in vivo to correct the inherited deficiency of bilirubin glucuronidation in Gunn rats. [3][4][5] However, the duration of expression of genes transferred by adenoviral vectors is limited because of the episomal nature of the virus. Furthermore, the host humoral and cellular immune response precludes gene expression following subsequent virus administration. 6-9 The anti-adenoviral response consists of both humoral and cell-mediated components, and both have been reported to contribute to the loss of expression of adenovirally delivered therapeutic genes. 9 Studies in nude and SCID mice with d...