Gene therapy for murine mucopolysaccharidosis type VII.

Mucopolysaccharidosis type VII (MPS VII) is caused by a deficiency in the lysosomal enzyme beta-glucuronidase resulting in the accumulation of undegraded glycosaminoglycans in many tissues. A murine model of MPS VII shares many of the clinical, biochemical and histopathological features of human MPS VII and has provided an opportunity to study novel therapeutic approaches in a system with a uniform genetic background. Retroviral mediated gene therapy directed to the hematopoietic system or to artificial neo-organs resulted in low levels of enzyme in several tissues and reduced lysosomal storage in the liver and spleen. Partial correction of the disease in the eye was observed following an intravitreal injection of recombinant adenovirus. Neither retroviral nor adenoviral mediated gene transfer techniques resulted in a systemic reduction of lysosomal storage. Here we discuss several novel gene transfer approaches designed to increase the systemic levels of beta-glucuronidase in the MPS VII mouse.

Embryonic stem cell lines derived from blastocysts by a simplified technique.

We have isolated euploid, pluripotent stem cell lines directly from mouse blastocysts by a simple culture technique. Our method permits cell lines to be derived from individual embryos, without the use of ovariectomy, immunosurgery and conditioned medium. We cultured individual intact blastocysts in MicroTest plates on top of a feeder layer of lethally irradiated STO mouse fibroblasts in a 10-microliters volume of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The cell lines maintain a stable complement of 40 chromosomes, form embryoid bodies and differentiate both in culture and in solid tumors.

Altered trophoblast functions in implantation-defective mouse embryos.

Extracellular proteases, such as plasminogen activator (PA), may play a role in the invasive action of trophoblasts during blastocyst implantation in mice. Detailed analysis of proteases released by trophoblasts must be carried out in culture for practical reasons, but the in vitro results should ideally be related to implantation in utero to test the validity of the conclusions in the model system. The implantation-defective mutant (tw73) allows us to investigate alterations of trophoblasts in culture which will reflect their altered functions in utero. The trophoblasts of mutant embryos attached and proliferated in culture to the same extent as control embryos. Therefore, the mutant effect is not to kill or stop the division of these cells. However, trophoblasts of tw73 homozygotes showed decreased invasive ability when transplanted into testes. To examine the biochemical basis of altered invasive behavior in mutants, PA activity released by blastocysts in culture was measured. Medium conditioned by tw73 homozygotes contained less PA fibrinolytic activity than culture medium of control embryos. The correlation between decreased in vitro PA activity and the invasive defects seen in utero and ectopically, supports the involvement of PA in the invasive phase of the implantation process.

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines.

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.

Modification of monoclonal antibody carbohydrates by oxidation, conjugation, or deoxymannojirimycin does not interfere with antibody effector functions.

Site-specific attachment of metal chelators or cytotoxic agents to the carbohydrate region of monoclonal antibodies results in clinically useful immunoconjugates [Doerr et al. (1991) Ann Surg 214: 118, Wynant et al. (1991) Prostate 18: 229]. Since the capacity of monoclonal antibodies (mAb) to mediate tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may accentuate the therapeutic effectiveness of immunoconjugates, we determined whether site-specific modification of mAb carbohydrates interfered with these functions. The chemical modifications examined consisted of periodate oxidation and subsequent conjugation to either a peptide linker/chelator (GYK-DTPA) or a cytotoxic drug (doxorubicin adipic dihydrazide). mAb-associated carbohydrates were also modified metabolically by incubating hybridoma cells in the presence of a glucosidase inhibitor deoxymannojirimycin to produce high-mannose antibody. All four forms (unaltered, oxidized, conjugated and high-mannose) of murine mAb OVB-3 mediated tumor cell lysis via CDC. Similarly, equivalent ADCC was observed with native and conjugated forms of mAb OVB-3 and EGFR.1. ADCC was achieved with different murine effector cells such as naive (NS), poly (I*C)- and lipopolysaccharide-stimulated (SS) spleen cells, or Corynebacterium-parvum-elicited peritoneal cells (PEC). All murine effector cell types mediated tumor cell lysis but differed in potency such that PEC > SS > NS. Excellent ADCC activity was also demonstrable by human peripheral blood mononuclear cells with OVB-3-GYK-DTPA and high-mannose OVB-3 mAb. ADCC activity was detectable in vivo: both native and conjugated OVB-3 inhibited growth of OVCAR-3 xenografts in nude mice primed with C. parvum. In conclusion, modification of mAb carbohydrates did not compromise their in vivo or in vitro biological functions. Therefore, combination therapy using immunomodulators to enhance the effector functions of site-specific immunoconjugates could be seriously contemplated.

Trace amounts of murine immunoglobulin in affinity purified leukocyte interferon alpha are not immunogenic.

Human leukocyte-derived interferon alfa-n3 (Alferon N Injection) is purified to very high specific activity over a murine immunoaffinity column specific for human interferon alpha. Trace amounts of murine immunoglobulin copurify with the interferon alfa-n3. Three populations of individuals were studied for the development of human anto-murine antibodies (HAMA), that is, normal donors, Condylomata acuminata patients receiving interferon alfa-n3, and Condylomata acuminata patients receiving placebo. High and variable endogenous levels of HAMA were observed in all three populations. The same relative increase in HAMA was seen in the placebo as in the interferon alfa-n3 treatment groups. The data demonstrate that intralesional injection of the interferon alfa-n3 did not induce the development of HAMA.

Absence of neutralizing antibodies to interferon in condyloma acuminata and cancer patients treated with natural human leukocyte interferon.

Human leukocyte-derived interferon-alpha (IFN-alpha n3) was used to treat condyloma acuminata patients in a double-blind placebo-controlled clinical study. The incidence of antibody formation to IFN-alpha was evaluated in matched patient sera from the control placebo and the IFN-alpha n3 treatment groups. Sera from IFN-alpha n3-treated phase I cancer patients and untreated healthy donors were also evaluated. Three sensitive assay methods (ELISA, competitive immunoradiometric, and antiviral neutralization) were used in these evaluations. The overall levels of detectable binding anti-IFN-alpha antibodies in the patients were similar to those of the normal donors. No neutralizing antibodies were generated in the patients after repeated treatment with natural IFN-alpha n3.

Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase.

Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.

Design of compounds that increase the absorption of polar molecules.

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Intestinal transport of gentamicin with a novel, glycosteroid drug transport agent.

PURPOSE: The objective was to investigate the ability of a glycosteroid (TC002) to increase the oral bioavailability of gentamicin. METHODS: Admixtures of gentamicin and TC002 were administered to the rat ileum by injection and to dogs by ileal or jejunal externalized ports, or PO. Bioavailability of gentamicin was determined by HPLC. 3H-TC002 was injected via externalized cannulas into rat ileum or jejunum, or PO and its distribution and elimination was determined. The metabolism of TC002 in rats was evaluated by solid phase extraction and HPLC analysis of plasma, urine and feces following oral or intestinal administration. RESULTS: The bioavailability of gentamicin was substantially increased in the presence of TC002 in both rats and dogs. The level of absorption was dependent on the concentration of TC002 and site of administration. Greatest absorption occurred following ileal orjejunal administration. TC002 was significantly more efficacious than sodium taurocholate, but similar in cytotoxicity. TC002 remained primarily in the GI tract following oral or intestinal administration and cleared rapidly from the body. It was only partly metabolized in the GI tract, but was rapidly and completely converted to its metabolite in plasma and urine. CONCLUSIONS: TC002 shows promise as a new drug transport agent for promoting intestinal absorption of polar molecules such as gentamicin.

Cationic facial amphiphiles: a promising class of transfection agents.

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.