Strategy to detect pre-existing immunity to AAV gene therapy.

Gene therapy may offer a new treatment option, particularly for patients with severe hemophilia, based on recent research. However, individuals with pre-existing immunity to adeno-associated viruses (AAVs) may be less likely to benefit from AAV vector-based therapies. To study pre-existing AAV5 immunity in humans, we validated two complementary, sensitive, and scalable in vitro assays to detect AAV5 total antibodies and transduction inhibition (TI). Using these two assays, we found that 53% of samples from 100 healthy male individuals were negative in both assays, 18% were positive in both assays, 5% were positive for total antibodies but negative for TI and, of interest, 24% were negative for total antibodies but positive for TI activity, suggesting the presence of non-antibody-based neutralizing factors in human plasma. Similar findings were obtained with 24 samples from individuals with hemophilia A. On the basis of these results, we describe the development of a dual-assay strategy to identify individuals without total AAV5 antibodies or neutralizing factors who may be more likely to respond to AAV5-directed gene therapy. These assays offer a universal, transferrable platform across laboratories to assess the global prevalence of AAV5 antibodies and neutralizing factors in large patient populations to help inform clinical development strategies.

Stable expression of rat dihydrodiol dehydrogenase (AKR1C9) in human breast MCF-7 cells results in the formation of PAH-o-quinones and enzyme mediated cell death.

Dihydrodiol dehydrogenase members of the aldo-keto reductase (AKR) superfamily have been implicated in the metabolic activation of PAH trans-dihydrodiols because they convert these proximate carcinogens to reactive and redox-active o-quinones. In this study, rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MCF-7) cells, which represent a null-environment for AKR expression, to detect the formation of PAH o-quinones in a cellular context and the cellular consequences of o-quinone formation. The heterologous transfected cells expressed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indicated that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that observed in rat liver cytosol. The transfectants were found to convert (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ). The identity of this metabolite was confirmed by co-chromatography and by UV-Vis diode-array spectrometry. This conversion was not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 microM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate dehydrogenase (LDH) release, occurred over the time course of o-quinone formation leading to 77% of the cellular LDH being released by 16 h. AKR1C9 inhibitors blocked the B[a]P-7,8-diol dependent cytotoxicity indicating that it was mediated by the enzymatically formed BPQ. These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity due to the formation of unconjugated BPQ.

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Metabolism of [14C]naphthalene in the B6C3F1 murine isolated perfused liver.

Naphthalene (NA) is metabolized by pulmonary and hepatic tissues to epoxides, quinones, and their related phase II metabolites. To delineate specific liver metabolites, metabolism was studied in B6C3F1 mouse liver perfused with 5 and 10 mumol/hr [14C]NA. Liver metabolites were compared with urinary metabolites from mice exposed to an equivalent total dose of NA (50 mg/kg ip) to ascertain interorgan and extrahepatic transformation in vivo. Metabolites were separated into pools via hydrophobic columns under neutral (pool I) and acid (pool II) conditions. Pool I contained the majority of [14C] in perfusate and urine. In perfusate, high levels of sulfate conjugates of naphthol and dihydroxynaphthalene were found along with dihydrodiol and glucuronic acid conjugates. In the urine, dihydrodiol was the most abundant metabolite. A novel N-acetylated glutathione conjugate was a constituent of pool II of both perfusate and urine. Additional metabolites identified in urine were N-acetylcysteine conjugate of dihydrodiol epoxide, mercaptolactic acid conjugate of naphthalene oxide (NO), and diglucuronide and sulfate/glucuronide conjugates of dihydroxynaphthalene. Mercapturic acid conjugates of NO were not observed in either perfusate or urine; this finding highlights metabolic differences between strains. Differential covalent binding occurred in cellular fractions, with the highest binding occurring in microsomes and mitochondria. These metabolites indicate that interorgan metabolism plays a role in the disposition of NA in vivo.

Formation of epoxide and quinone protein adducts in B6C3F1 mice treated with naphthalene, sulfate conjugate of 1,4-dihydroxynaphthalene and 1,4-naphthoquinone.

Naphthalene (NA) is metabolically activated to the reactive intermediates, naphthalene oxide (NO) and naphthoquinones. To investigate the role of circulating reactive metabolites in NA toxicity, the half-life of NO was examined. The in vitro half-life of NO in both whole blood and plasma was 10 min. Detectable levels of NO were seen in perfusate leaving the isolated perfused liver of B6C3F1 mice infused with 10 mumol/h NA. Identification of protein sulfhydryl adducts in NA-exposed mice (50 and 100 mg/kg, IP, 24 h) revealed a predominance of quinone adducts in liver, lung, kidney, red blood cells and brain. The epoxide adduct predominated in plasma protein. Administration of the sulfate conjugate of 1,4-dihydroxynaphthalene (NHQS) (100 mg/kg) resulted in formation of naphthoquinone protein sulfhydryl adducts in lung, liver and kidney. Administration of 1,4-naphthoquinone (NQ) (5 mg/kg) produced NQ adducts in liver, lung, kidney, plasma and brain.