The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype.

Although the latent membrane protein-1 (LMP1) of the Epstein-Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non-viral vector-based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkin's lymphoma cell lines. Strikingly, LMP1 down-regulated the expression of B-cell-specific genes including B-cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B-cell differentiation. Our data suggest that in EBV-positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed-Sternberg cells, including the loss of B-cell identity.

Comparative analysis of the expression of the Epstein-Barr virus (EBV) anti-apoptotic gene BHRF1 in nasopharyngeal carcinoma and EBV-related lymphoid diseases.

Epstein-Barr virus (EBV) has been identified in a wide range of neoplastic and non-neoplastic disorders. The EBV open reading frame BHRF1 encodes a protein with partial sequence and functional homology to the anti-apoptotic onco-protein Bcl-2 and may therefore have a role in the proliferation of EBV positive cells. We have developed a rat monoclonal antibody against pBHRF1, which can detect BHRF1 in paraffin sections. While a number of mutant versions of BHRF1 were recognised, the monoclonal did not detect the BHRF1 homologue encoded by Herpesvirus papio or two mutants with deletions in the BH2 region. This novel rat monoclonal antibody (6A9) was used to examine tissue sections from 39 cases of non-keratinising undifferentiated nasopharyngeal carcinoma (NPC), 6 cases of metastatic NPC, 7 cases of EBV-positive NPC with squamous differentiation from Chinese patients, 15 cases of EBV-positive post-transplant lymphoproliferative disorder (PTLD), 6 EBV-containing lymphoblastoid cell lines, and 2 cases of oral hairy leukoplakia (OHL). In 11 cases of undifferentiated NPC, RT-PCR data were available for comparison with the immunohistochemistry. Both cases of OHL and two cases of LCL were positive for BHRF1 but none of the PTLD showed positive staining. All cases of undifferentiated NPC were positive for Bcl-2 but only one BHRF1 positive cell was identified in 1 of 39 cases of primary undifferentiated NPC. The 6A9 antibody produced less background staining and no nuclear positivity compared with the commercially available mouse monoclonal 5B11. It is concluded that BHRF1 can not be detected by immunohistochemistry in NPC and therefore it appears not to play a significant anti-apoptotic role in the progression of this EBV-associated tumour. The 6A9 monoclonal appears to be superior to 5B11 for the detection of pBHRF1 in tissue sections.

Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma.

Patients suffering from nasopharyngeal carcinoma (NPC) generally exhibit elevated serum IgA antibody titres to Epstein-Barr virus (EBV) early antigen (EA) and virus capsid antigen (VCA). This property is frequently used as a diagnostic aid. Preliminary experiments suggested that an ELISA for IgA antibodies against the EBV-encoded thymidine kinase (TK) could form the basis of a more reliable diagnostic test. Here, we describe the construction of a recombinant baculovirus that expresses the EBV TK and present a full analysis of its use in serological surveys of NPC patients. Baculovirus-derived TK was used to develop a simple ELISA for serum IgA against this antigen. ELISA reactivity was strongly associated with NPC compared with an EBV-positive, normal control population. Comparison with the existing IgA-VCA and EA assays showed that the TK ELISA had higher sensitivity whilst the specificity was similar or higher. We conclude that the TK ELISA presents a strong predictor of NPC and, in its refined form, has improved pickup rates. In addition, results from patients with chronic nasopharyngitis (CNP) suggest that individuals with both symptoms of CNP and an elevated TK ELISA value may be at increased risk for the development of head-and-neck cancer.

Heterogeneity of HLA and EBER expression in Epstein-Barr virus-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an aggressive tumour of multifactorial aetiology that, although rare in most parts of the world, poses a significant mortality problem in its high incidence area of Southern China. Improved therapies are an urgent requirement and, towards this end, immunotherapeutic methods are being developed in several centres. Such strategies are dependent on the immune competence of the target tumour, in particular its expression of HLA class-I. We examined HLA class-I and -II expression in 27 primary NPC biopsies and found that 15% were extensively down-regulated for class-I expression with the majority of tumour cells appearing negative. Whilst HLA class-II was expressed at high levels in the majority of tumours, 37% showed substantial down-regulation. NPC is associated with Epstein-Barr virus (EBV). Expression of the virus-encoded EBER RNAs is accepted as a marker of EBV latency and is regarded as a valuable diagnostic criterion. EBER RNAs were expressed in all samples, but in some the level was remarkably heterogeneous, being barely detectable in many tumour cells. Our study reinforces the concept of extensive phenotypic variation in NPC. There are morphological differences between tumour cells. Some tumours express HLA class-I and/or -II, whilst others are down-regulated or negative. Individual tumours may or may not express the EBV-encoded LMP-1 protein, and individual tumour cells may express high levels of EBER, yet adjacent tumour cells express very little or none.

Heparin and heparan sulfate bind interleukin-10 and modulate its activity.

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-beta, IL-7, and interferon-gamma. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin-agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, K(d), of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10-induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC(50) values of 100 to 500 microg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC(50) of 2,000 to 5,000 microg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10- induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10-mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity. (Blood. 2000;96:1879-1888)

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA.

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

Herpesvirus papio encodes a functional homologue of the Epstein-Barr virus apoptosis suppressor, BHRF1.

The human tumour virus Epstein-Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the BCL:-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.

Epidemiology of herpesvirus papio infection in a large captive baboon colony: similarities to Epstein-Barr virus infection in humans.

The epidemiology of herpesvirus papio, a lymphocryptovirus similar to Epstein-Barr virus (EBV), was studied in a captive colony of >1900 baboons. Herpesvirus papio IgG antibody titers were measured by IFA. In total, 438 specimens from 296 baboons were assessed, including 116 serial specimens from 52 juveniles and 6 infants studied monthly for 1 year following birth and at age 18 months. Maternally derived antibody reached a nadir at 4 months of age. About 75% of animals at 12 months of age and >95% of animals after age 24 months demonstrated serologic evidence of herpesvirus papio infection. After age 3 years, the geometric mean titer was 1:60-75. The epidemiology of herpesvirus papio infection in baboons closely parallels that of EBV infection in humans. An animal model of lymphocryptovirus infection will facilitate investigations of human lymphocryptovirus biology.

Antigen gene transfer to cultured human dendritic cells using recombinant avipoxvirus vectors.

Advances in understanding the role of dendritic cells (DCs) as the major antigen (Ag)-presenting cell type of the immune system combined with the recent development of methods for the ex vivo expansion of human DCs have opened the possibility for the transfer of tumor Ags to DCs with a view toward tumor immunotherapy. In this study, we examined the feasibility of Ag transfer to cultured human DCs using the host range-restricted avipoxvirus, fowlpoxvirus (FWPV). FWPV was found to infect and express a lacZ marker gene in a number of mammalian cell lines of fibroblastic, epithelial, and hemopoietic lineage origins. LacZ recombinant FWPV (rFWPV) was found subsequently to infect human DCs that had been cultured ex vivo from peripheral blood monocytes. Using rFWPV containing lacZ under the control of a vaccinia virus (VV) early/late promoter (p7.5K) and a 10 plaque-forming units per cell multiplicity of infection, >80% of cells expressed the lacZ marker gene. Quantitative analysis showed that the level of expression continued to rise for 5 days postinfection, at which point the experiments were terminated. Replication-competent recombinant VV (rVV) was also shown to be capable of transferring the marker gene to primary DC cultures. However, neither rFWPV nor rVV were able to express transgenes under the control of late viral promoters, indicating that both rFWPV and rVV infections are arrested at an early stage in human DCs. Infection of CD83 + DCs by rFWPV was confirmed by double-staining cytochemistry. We conclude that host range-restricted FWPV can be used efficiently to transfer Ag genes to human DCs ex vivo and may have a role in the development of tumor immunotherapy protocols.

Recombinant adeno-associated virus-mediated expression of O6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity.

Recombinant adeno-associated virus (rAAV) encoding the human O6-alkylguanine-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeLa clones were isolated, 9 of which survived for 2-3 months before cell death ensued, presumably owing to the loss of G418 resistance. Northern blot analysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced from this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg protein was expressed relative to untransduced cells (800-900 fmol/mg protein). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold higher than background conferred significant resistance (p < 0.001) to the toxic effects of this drug. Two days following infection of K562 cells with the rAAV vector, immunoblot analysis showed that hAT protein was being produced. Three K562 clones, isolated using G418 selection, were studied in detail and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 cells contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot analysis showed the production of an appropriately sized transcript and immunoblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Expression of hAT at levels 800- to 1500-fold higher than background conferred significant resistance (p < 0.001) to the toxic effects of mitozolomide. We have therefore successfully conferred a protective advantage against mitozolomide toxicity to cells by rAAV-mediated hAT expression.

Immunization of common marmosets with Epstein-Barr virus (EBV) envelope glycoprotein gp340: effect on viral shedding following EBV challenge.

Epstein-Barr virus (EBV), the cause of infectious mononucleosis, is involved in the pathogenesis of several human cancers, the highest frequency of association being found in undifferentiated nasopharyngeal carcinoma and endemic Burkitt's lymphoma. The development of animal models in which potential vaccines can be tested is important. EBV infection of the common marmoset, using the M81 strain originally derived from a patient with nasopharyngeal carcinoma, induces a carrier state in this animal. Persistent infection is characterized by the production of antibodies to viral antigens, and the secretion of EBV DNA into buccal fluids. Following immunization with envelope glycoprotein gp340 derived from a bovine papilloma virus expression vector, prior to EBV infection, viral DNA was detected significantly less frequently in the buccal fluids of immunized, than of nonimmunized, infected animals, indicating that although the carrier state had not been abolished, it had been altered. A reduction in virus load was also observed when offspring of seronegative, and on occasion seropositive, parents were immunized neonatally, before EBV challenge.

The processing, transport and heterologous expression of Epstein-Barr virus gp110.

Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.

Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.

Towards gene therapy of Hurler syndrome.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler's syndrome. However, due to a lack of matched related donors and unacceptable morbidity of matched unrelated transplants, this therapy is not available to all patients. Therefore we have been developing an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. A number of different gene transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of haemopoietic colonies as around 50%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. Indeed a possible disadvantage has been identified for the use of intensive transduction procedures. The enzyme is secreted into the medium and functional localisation has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This pre-clinical work forms the basis for a clinical trial of gene therapy for Hurler syndrome.

Differences in serological IgA responses to recombinant baculovirus-derived human papillomavirus E2 protein in the natural history of cervical neoplasia.

Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Epstein-Barr virus associated graft failure following heart/lung transplantation.

A case is described of late pulmonary graft failure in a heart/lung transplant recipient. The major characteristics were alveolar fibrosis and a restrictive physiological deficit. Epstein-Barr virus was implicated as an aetiological agent using immunohistochemical analysis and by a response to treatment with ganciclovir.

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.

Murine gammaherpesvirus-68 encodes homologues of thymidine kinase and glycoprotein H: sequence, expression, and characterization of pyrimidine kinase activity.

We have sequenced a 4.5-kb fragment of DNA spanning the junction of the BamHI D and E fragments of murine gammaherpesvirus-68 (MHV-68). This sequence was found to code for two major open reading frames (orfs) of 1934 and 2192 bp which showed significant homology to the thymidine kinase (TK) and glycoprotein H (gH) sequences of other gammaherpesviruses. Upstream from the TK gene another orf was found which showed amino acid sequence homology to the HSV1 UL24 gene. Analysis of the 1934-bp orf revealed the presence of all six of the recognized sites that are conserved between herpesvirus TKs although, uniquely among sequenced herpesvirus TK enzymes, MHV-68 lacks the consensus nucleotide binding site GXXGXGK, the second glycine being replaced by alanine. The MHV-68 TK has a predicted M(r) of 68,443, while the gH is predicted to have a M(r) of 82,890. Northern blot analysis showed an early TK message of 2.6 kb and a late gH-specific message of 2.5 kb. Both TK and gH probes detected a 4.3-kb late message, implying that this late message spans gH and TK. The TK coding sequence was expressed using an in vitro transcription translation system and was shown to encode functional TK activity.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.