Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics:  a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES)

Previous studies have documented that 0.1Ϸ1% of input recombinant adeno-associated virus (rAAV) vectors could be stabilized and lead to transgene expression. To characterize the steps involving massive AAV DNA loss, we designed an''AAV footprinting'' strategy that can track newly formed AAV dsDNA genomes. This strategy is based on an ROSA26R mouse model or cell line that carries a lacZ gene flanked by two loxP sites. When it is transduced by a rAAV vector carrying the Cre recombinase, the lacZ gene can be activated and remain active even when rAAV genomes are later lost. By using this sensitive AAV footprinting technique, we confirmed the existence of transient AAV dsDNA that went undetected by conventional DNA methods. Although these dsDNA intermediates could be efficiently formed in almost every cell and were competent for mRNA transcription and protein synthesis in vivo, they got lost continuously. Only a small fraction was eventually stabilized for sustained gene expression. Although both rAAV2 and rAAV8 can potentially have similar levels of dsDNA formation, AAV8 dsDNA was formed much faster than that of AAV2, which explains why rAAV8 is more efficient than rAAV2 in transducing the liver. Collectively, our studies suggested that rather than receptor binding, viral entry, and ssDNA to dsDNA conversion, the instability of newly formed AAV dsDNA was the primary contributing factor for the low rAAV transduction efficacy. The uncoating step significantly influenced the stability of AAV transient dsDNA. The identification of transient AAV dsDNA provided a new pathway for improving rAAV transduction. adeno-associated virus ͉ DNA stability ͉ gene therapy ͉ vector ͉ ROSA26R

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10-100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a ␤-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3-to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3-to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a “chain imbalance” problem in which LC secretion is ~1–2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC745). The optimized HC mutein, HCHL, including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HCHL+AAV8-LC or AAV8-HC745+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HCHL+AAV8-LC achieved three- to four-fold higher HC expression than AAV8-HC745+AAV8-LC, consistent with the FVIII functional assays. HCHL should be substituted for HC745 in a dual AAV vector strategy due to its enhanced expression.

No abstract

We found that the endogenous ERPs (P3 and N2) were significantly affected in children with ADHD, compared to exogenous ERPs (N1 and P2). Increased latency of P3 suggests a slower processing speed, and decreased P3 amplitude is interpreted as disruption of inhibitory control in children with ADHD. These results indicate a neurocognitive abnormality in ADHD, as presented by a reduction in ERP response.