Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

Resection upregulates the IGF-I system of parenterally fed rats with jejunocolic anastomosis.

Rats maintained with parenteral nutrition following 60% jejunoileal resection plus cecectomy exhibit minimal adaptive growth in the residual jejunum but a dramatic adaptive growth in the residual colon. Coinfusion of insulin-like growth factor I (IGF-I) with parenteral nutrition induces jejunal growth but has minimal effects in the colon. Our objective was to study the role of the endogenous IGF-I system in the differential responses of jejunum and colon to resection and/or IGF-I during parenteral nutrition. We measured concentrations of immunoreactive IGF-I in plasma, jejunum, and colon, IGF-I receptor binding, and levels of IGF receptor, IGF-I, IGF binding protein (IGFBP)-3 and IGFBP-5 mRNA in residual jejunum and colon 7 days after resection and/or IGF-I treatment. IGF-I receptor number was increased (74-99%) in jejunum and colon due to resection; IGF-I mRNA was increased 5-fold in jejunum and 15-fold in colon due to resection. Resection increased circulating IGFBPs but did not alter plasma IGF-I concentration. Resection induced colonic growth in association with significantly greater colonic IGFBP-5 mRNA and significantly lower colonic immunoreactive IGF-I. IGF-I treatment had no significant effect on IGF-I mRNA or IGF-I receptor number. Concentrations of plasma and jejunal immunoreactive IGF-I were significantly increased in rats given IGF-I in association with jejunal growth. IGF-I treatment significantly increased IGFBP-5 mRNA in the jejunum, which also correlated with jejunal growth. Thus resection upregulated IGF-I receptor number and IGF-I mRNA in residual jejunum and colon, but differential adaptation of these segments correlated with differential regulation of IGFBP-5 mRNA.

A comparison of two opioid analgesics for relief of visceral pain induced by intestinal resection in rats.

While developing a rat model for human short bowel syndrome, we noted that untreated rats as well as rats administered buprenorphine after intestinal resection exhibited behavior and appearance consistent with visceral pain and distress. To provide appropriate analgesics, we developed criteria to assess pain-related behavioral changes and conducted an experiment to evaluate the effectiveness of buprenorphine versus oxymorphone to alleviate the pain induced by intestinal resection. Rats underwent either small-bowel resection or transection surgery; in addition, animals received jugular catheterization for the delivery of total parenteral nutrition (TPN). Rats treated with buprenorphine received 0.5 mg/kg every 6 h subcutaneously, and rats treated with oxymorphone received 0.03 mg/kg hourly for 32 h via continuous intravenous (i.v.) infusion with TPN solution. Rats treated with buprenorphine exhibited behavior and appearance consistent with pain and distress for as long as 32 h postoperatively, whereas animals treated with oxymorphone exhibited behavior and appearance similar to their preoperative state. Thus, oxymorphone alleviated the pain-related behavioral changes after intestinal resection far better than did buprenorphine. Of interest, we observed that the buprenorphine was associated with a decrease in the volume of urine collected, whereas oxymorphone was associated with urine volumes similar to those of nonresected rats maintained with TPN. Because oxymorphone appeared to be a superior analgesic, we also evaluated three routes for administering this drug. Pain-related behavior changes were alleviated by the administration of oxymorphone by either Alzet mini-pump, bolus i.v. injection, or continuous i.v. infusion. We conclude that compared with buprenorphine, oxymorphone is a superior analgesic for the alleviation of visceral pain due to intestinal resection.

Enterotrophic effect of insulin-like growth factor-I but not growth hormone and localized expression of insulin-like growth factor-I, insulin-like growth factor binding protein-3 and -5 mRNAs in jejunum of parenterally fed rats.

BACKGROUND: Administration of insulin-like growth factor (IGF)-I, but not growth hormone (GH), stimulates mucosal hyperplasia in surgically stressed rats with intestinal atrophy induced by hypocaloric total parenteral nutrition (TPN). Our aim was to characterize the basis for this disparity in enterotrophic action by assessing the relationships between stimulation of intestinal growth, nutritional adequacy, and localization of expression of IGF-I, insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mRNAs in jejunum. METHODS: Rats were maintained with TPN for 8 days and treated with IGF-I or GH and adequate nutrition for 5 days after recovery from surgery. Jejunal mass, morphology, and sucrase activity were assessed. Localization of expression of IGF-I, IGFBP-3, and IGFBP-5 mRNAs in jejunum was accomplished by in situ hybridization. RESULTS: Serum IGF-I and body weight gain were significantly increased by IGF-I or GH. Jejunal mucosal dry mass, morphology, and sucrase activity were improved with IGF-I but not GH. There were no differences in IGF-I mRNA. IGFBP-3 mRNA was localized in the lamina propria of the villi. IGF-I or GH stimulated IGFBP-3 expression. IGF-I strongly stimulated IGFBP-5 expression in the lamina propria and the muscularis and induced a twofold increase in IGFBP-5 mRNA based on RNase protection assay of intact jejunum total RNA. GH induced a modest increase in IGFBP-5 expression in the muscularis with no effect on intact jejunum total RNA. CONCLUSIONS: The GH resistance observed in the jejunal mucosa of TPN rats cannot be fully explained by inadequate nutrition. The expression of IGFBP-5 in the lamina propria suggests it may modulate the enterotrophic action of exogeneous IGF-I.

Differential jejunal and colonic adaptation due to resection and IGF-I in parenterally fed rats.

Patients with severe short-bowel syndrome (SBS) often require long-term total parenteral nutrition (TPN) to maintain their nutritional status because of limited intestinal adaptation. Growth factors, including insulin-like growth factor I (IGF-I), are under investigation to promote intestinal adaptation and tolerance to oral feeding. We investigated structural and functional adaptation of the jejunum and colon in four groups of rats maintained with TPN for 7 days after a 60% jejunoileal resection and cecectomy or sham surgery and treatment with IGF-I or vehicle. Resection alone did not stimulate jejunal growth. IGF-I significantly increased jejunal mucosal mass, enterocyte proliferation, and migration rates. IGF-I decreased jejunal sucrase specific activity and reduced active ion transport and ionic permeability; resection alone had no effect. In contrast, resection significantly increased colonic mass and crypt depth but had no effect on active ion transport or ionic permeability. IGF-I had minimal effects on colonic structure. IGF-I but not resection stimulates jejunal adaptation, whereas resection but not IGF-I stimulates colonic growth in rats subjected to a model for human SBS. IGF-I treatment may improve intestinal adaptation in humans with SBS.

Investigation of insulin-like growth factor (IGF)-I and insulin receptor binding and expression in jejunum of parenterally fed rats treated with IGF-I or growth hormone.

To investigate the ability of insulin-like growth factor-I (IGF-I), but not GH, to stimulate jejunal growth, we compared indices of IGF-I and insulin receptor expression in jejunal membranes from rats maintained with total parenteral nutrition (TPN) and treated with rhIGF-I and/or rhGH. TPN without growth factor treatment (TPN control) induced jejunal atrophy, reduced serum IGF-I, increased serum insulin concentrations, and increased IGF-I receptor number, IGF-I receptor messenger RNA, and insulin-specific binding to 133% to 170% of the orally fed reference values, P < 0.01. Compared with TPN control, IGF-I or IGF-I + GH stimulated jejunal mucosal hyperplasia; IGF-I treatment increased serum IGF-I by 2- to 3-fold and decreased serum insulin concentrations by 60%, decreased IGF-I receptor number by 50% (P < 0.001), and increased insulin receptor affinity and insulin receptor protein content. Treatment with GH alone increased serum IGF-I concentration, did not alter TPN-induced jejunal atrophy, and decreased insulin-specific binding and insulin receptor protein content (39% and 59%, respectively, of the TPN control values, P < 0.01). We conclude that: 1) jejunal IGF-I receptor content reflects circulating concentration of ligand and is not limiting for jejunal growth; and 2) increased circulating concentration of IGF-I may promote jejunal growth via interaction with jejunal insulin or IGF-I receptors.