Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency.

Development of operational immunologic tolerance with neonatal gene transfer in nonhuman primates: preliminary studies.

Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses.

AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic mouse.

Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.

AAV-based neonatal gene therapy for hemophilia A: long-term correction and avoidance of immune responses in mice.

Hemophilia A gene therapy has been hampered by immune responses to vector-associated antigens and by neutralizing antibodies or inhibitors against the factor VIII (FVIII) protein; these 'inhibitors' more commonly affect hemophilia A patients than those with hemophilia B. A gene replacement strategy beginning in the neonatal period may avoid the development of these immune responses and lead to prolonged expression with correction of phenotype, thereby avoiding long-term consequences. A serotype rh10 adeno-associated virus (AAV) was developed splitting the FVIII coding sequence into heavy and light chains with the chicken ß-actin promoter/CMV enhancer for dual recombinant adeno-associated viral vector delivery. Virions of each FVIII chain were co-injected intravenously into mice on the second day of life. Mice express sustained levels of FVIII antigen ≥5% up to 22 months of life without development of antibodies against FVIII. Phenotypic correction was manifest in all AAV-FVIII-treated mice as demonstrated by functional assay and reduction in bleeding time. This study demonstrates the use of AAV in a gene replacement strategy in neonatal mice that establishes both long-term phenotypic correction of hemophilia A and lack of antibody development against FVIII in this disease model where AAV is administered shortly after birth. These studies support the consideration of gene replacement therapy for diseases that are diagnosed in utero or in the early neonatal period.

rPSGL-Ig for improvement of early liver allograft function: a double-blind, placebo-controlled, single-center phase II study.

The selectin antagonist known as recombinant P-selectin glycoprotein ligand IgG (rPSGL-Ig) blocks leukocyte adhesion and protects against transplantation ischemia reperfusion injury (IRI) in animal models. This randomized (1:1) single-center double-blind 47-patient phase 2 study with 6-month follow-up assessed rPSGL-Ig's safety and impact on early graft function at 1 mg/kg systemic dose with pretransplant allograft ex vivo treatment in deceased-donor liver transplant recipients. Safety was assessed in all patients, whereas efficacy was assessed in a prospectively defined per-protocol patient set (PP) by peak serum transaminase (TA) and bilirubin values, and normalization thereof. In PP patients, the incidence of poor early graft function (defined as peak TA >2500 U/L or bilirubin >10 mg/dL), average peak liver enzymes and bilirubin, normalization thereof and duration of primary and total hospitalization trended consistently lower in the rPSGL-Ig group compared to placebo. In patients with donor risk index above study-average, normalization of aspartate aminotransferase was significantly improved in the rPSGL-Ig group (p < 0.03). rPSGL-Ig treatment blunted postreperfusion induction versus placebo of IRI biomarker IP-10 (p < 0.1) and augmented cytoprotective IL-10 (p < 0.05). This is the first clinical trial of an adhesion molecule antagonist to demonstrate a beneficial effect on liver transplantation IRI and supported by therapeutic modulation of two hepatic IRI biomarkers.

Death from metastatic donor-derived ovarian cancer in a male kidney transplant recipient.

Posttransplant malignancy developing in an allograft is an uncommon complication of organ transplantation. The tumor may represent malignant transformation of donor or recipient cells that were previously normal, metastatic malignancy of recipient origin or malignancy transmitted from organ donor to recipient. Establishing the origin of the malignancy is critical to treatment algorithms. It is generally believed allograft removal and immunosuppression withdrawal will lead to resolution of transmitted malignancies in cases where the renal allograft is the origin. We report a male patient who developed metastatic ovarian malignancy secondary to donor transmission.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

Adenovirus-mediated gene transfer to the peritoneum and hepatic parenchyma of fetal mice in utero.

BACKGROUND: The development of effective gene transfer in utero will provide alternative approaches to the treatment of genetic disorders. For many disorders, the fetal liver and peritoneum are important target tissues. Our goals were to compare the tissue sites and duration of transferred gene expression after intraperitoneal (i.p.) or intrahepatic adenoviral-mediated gene transfer in utero in the developing murine fetus. METHODS: Day 15 CD-1 fetuses were injected intrahepatically or intraperitoneally with recombinant adenoviruses containing the luciferase or beta-galactosidase reporter gene. Tissue levels of luciferase were quantitated, or tissues were examined for X-gal staining. RESULTS: Luciferase expression was observed in multiple fetal tissues (including brain, intestine, liver, and lung) and persisted up to 32 days after intrahepatic delivery. Significant hepatic tropism was demonstrated. CONCLUSIONS: Intrahepatic and intraperitoneal injection in utero results in transduction of multiple tissues in the developing murine fetus. Transuterine injection of fetal mice via intrahepatic and intraperitoneal routes provides a valuable model for assessing the efficacy of gene delivery vectors in the prenatal treatment of genetic disorders. These studies demonstrate that hepatic and intraperitoneal gene transfer to the developing murine fetus is feasible and may provide therapeutic levels of proteins during fetal development.

Fetal pulmonary sequestration: a favorable congenital lung lesion.

OBJECTIVE: We reviewed the perinatal clinical course of prenatally diagnosed pulmonary sequestrations to determine the natural history of this anomaly. METHODS: From January 1992 to August 1998, 192 women were referred to the University of California, San Francisco, Fetal Treatment Center because obstetric ultrasound had demonstrated fetal lung lesions. In 14 fetuses, the echogenic lung masses were pulmonary sequestrations deriving arterial blood supply from clearly identifiable systemic arteries rather than the pulmonary artery. We examined records of the 14 fetuses and documented the location of the lesion, gestational age at diagnosis, need for fetal intervention, prenatal and postnatal complications, gestational age at delivery, and survival. RESULTS: There were 16 intrathoracic pulmonary sequestrations in 14 fetuses (eight left-sided, four right-sided, two bilateral). Three fetuses had histologically mixed lesions (congenital cystic adenomatoid malformation and pulmonary sequestration). The mean age at diagnosis was 23 weeks' gestation (range 19-31 weeks). Two fetuses required prenatal intervention (placement of a thoracoamniotic shunt for drainage of an ipsilateral tension hydrothorax). The mean gestational age at delivery was 37 weeks (range 32-40 weeks). The large lesions of four fetuses regressed completely prior to birth, and the lesions of the remaining ten fetuses were electively resected after birth without causing morbidity or mortality. CONCLUSION: Pulmonary sequestrations are a subgroup of congenital lung lesions with a favorable outlook; many regress prenatally, and the persistent ones are resected safely postnatally. Pulmonary sequestrations cause hydrops only because of a tension hydrothorax, which can be drained prenatally, if necessary.

Evaluation of four new carbocyanine dyes for photodynamic therapy with lasers.

The search for improved photosensitizers for laser phototherapy of malignancies has led to the examination of a new group of carbocyanine dyes as effective fluorochromes. In this study, four carbocyanine dyes with different absorption maxima of 483 nm [DiOC6(3)], 545.5 nm (DiIC5(3)], 556.6 nm [DiSC5(3)], and 651.0 nm [DiSC3(5)] were tested in vitro. The kinetics of uptake and toxicity of these four dyes were assessed for P3 human squamous cell carcinoma, HT29 colon carcinoma, M26 melanoma, and TE671 fibrosarcoma cell lines at 15, 30, 45, 60, and 180 minutes after exposure with each dye. After sensitization with DiOC6(3), the P3 and M26 cell lines were also tested for phototherapy by treatment with 488-nm light from an argon laser. The results showed that these four carbocyanine dyes had rapid and significant uptake by the carcinoma cell lines with no toxicity at concentrations < 0.1 micrograms/mL. Nontoxic DiOC6(3) levels in sensitized tumor cells after laser phototherapy resulted in approximately 85% inhibition of P3 and approximately 95% inhibition of M26 cell lines by MTT assays. The results suggest that these carbocyanine dyes can be used for tumor photosensitization and wavelength-matched laser photodynamic therapy. Further in vivo studies will be necessary to define the clinical potential of carbocyanine dyes as tumor-targeting agents for phototherapy of cancer.

Polyomavirus-associated nephropathy in simultaneous kidney-pancreas transplant recipients: a single-center experience.

BACKGROUND: With the introduction of more potent immunosuppressive agents, rejection rates have decreased markedly in simultaneous pancreas-kidney transplant (SPK) recipients. However, with more intense immunosuppression, opportunistic infections such as polyoma virus have been more frequent. The purpose of this article is to outline the clinical course of SPK patients who developed documented polyoma infection in the transplanted kidney. METHODS: A retrospective review of 146 consecutive SPK recipients from 1996 to 2002 was performed. Induction and maintenance immunosuppression, surgical complications, rejection episodes, and opportunistic infections were reviewed. Patients who developed biopsy-proven polyoma virus infection in the renal allograft were identified. RESULTS: Nine patients (6%) were identified who developed polyoma. All had received induction therapy with either OKT3 (5 mg/d for 10.5 days) or thymoglobulin (5.7 mg/kg). Patients without polyoma had received similar induction. Maintenance immunosuppression included Prograf/MMF in six patients, CsA/MMF in two, and CsA/azathioprine in one. Time to diagnosis was an average of 359.3 days (range 136 to 836) after transplantation. Two patients had undergone treatment for kidney rejection prior to the diagnosis of polyoma. Immunosuppression was decreased in all patients when polyoma was identified, and more recently Cidofovir has been administered. Despite these interventions, five of the nine lost kidney function (creatinine > 5.0 or resumption of dialysis). However, none of the nine developed pancreatic abnormalities as demonstrated by normal blood glucose and amylase and no requirement for exogenous insulin. Two patients underwent LRRT more than 1 year after polyoma diagnosis; both have normal kidney function (Cr < 1.5 mg/dL) at 4 years of follow-up. Polyoma virus was the leading cause of renal loss in this cohort of patients. CONCLUSIONS: Polyoma is a serious concern for SPK transplant recipients. The pancreas, however, is spared from clinical evidence of infection, and no rejection was noted when immunosuppression was decreased. These graft losses appear to be a penalty of more potent immunosuppression, and a better treatment strategy is needed to prevent renal graft loss when polyoma is diagnosed. Retransplantation can be considered based on our limited experience.

Reconstruction of Tessier no. 4 clefts revisited.

Atypical craniofacial clefts present a surgical challenge. Little is published about their treatment because of their rarity. Such is the nature of Tessier no. 4 clefts and others with shortened oculoalar and oculo-oral distances requiring multiple operative interventions. The soft-tissue abnormalities include a cleft of the lip with possible extension into the check, orbital dystopia, and globe abnormalities and a decreased oculo-oral distance. Typically, multiple local cheek and nasal flaps are used to close the soft-tissue defects, elevate the medial canthus, and protect the globe. However, this approach creates traction on the lower eyelid, suboptimal location of the scar, and poor color match between the flaps. We present two cases of Tessier no. 4 clefts and one case of a multiply clefted (Tessier nos. 1/13, 2/12, 3/11) child with the typical contracted oculoalar and oculo-oral distances. Reconstruction with a superiorly based nasolabial flap transposed 90 degrees under the eye was performed in all three as a primary procedure. This technique respects aesthetic units and places the resulting scars in less conspicuous areas on the face.

A new nerve pedicle for finger sensibility: the dorsal digital sensory nerve.

Restoration of sensibility to the traumatized finger can be a difficult problem. Two patients with insensibility to the volar distal finger after trauma underwent delayed digital nerve repair. In the first patient, the dorsal branch of the radial proper digital nerve was approximated to the distal stump as a pedicle to span a 12-mm gap resulting from neuroma excision. The second patient had a 14-mm defect after scar-tissue excision 8 months following primary neurorrhaphy after trauma. Reconstruction was performed by approximating the dorsal branch of the radial proper digital nerve to the distal stump. Both patients had fingertip sensibility restored 1 year postoperatively, as documented by two-point discrimination. Anatomic dissections of 12 fresh cadaver fingers revealed a consistent pattern. Of the 24 proper digital nerves dissected, 23 had a distal dorsal sensory branch arising at the midportion of the proximal phalanx. The dorsal branch-vascularized pedicle of the proper digital nerve has not been described previously as a method for restoring finger sensibility in cases not amenable to primary neurorrhaphy. We believe this technique should be added to the repertoire of the practicing hand surgeon.

A strategy for primary reconstruction of long gap esophageal atresia using neonatal colon esophagoplasty: a case report.

Treatment options for long gap esophageal atresia without tracheoesophageal fistula generally require several stages over many months. An early neonatal vascularized conduit would allow a tension-free anastomosis, but the precarious blood supply of the neonatal bowel makes mobilization and immediate interposition hazardous. This report describes the successful application of a strategy for primary reconstruction in the neonate using a short piece of colon mobilized into the mediastinum for subsequent delayed anastomosis.

Anterior cervical approach for repair of laryngotracheoesophageal cleft.

Laryngotracheoesophageal cleft is a very rare congenital malformation characterized by a long midline communication between the esophagus and trachea extending from the larynx down the trachea for a variable distance. The most common repair strategy involves separation of the trachea and esophagus by a lateral approach via both a thoracotomy and a neck incision. The authors report a case of complete repair (without thoracotomy) via a neck incision through the anterior wall of the trachea.

Prospective analysis of lung-to-head ratio predicts survival for patients with prenatally diagnosed congenital diaphragmatic hernia.

BACKGROUND: Accurate prenatal prediction of outcome for fetuses who have congenital diaphragmatic hernia (CDH) is very difficult. The authors previously reported a retrospective analysis of risk factors for fetal CDH and proposed a new index of severity: the lung-to-head ratio (LHR). The authors now report a prospective study to test whether this new index predicts neonatal outcome. METHODS: Fifteen patients who had left-sided CDH were sonographically evaluated at the University of California, San Francisco (UCSF) and followed prenatally and postnatally. LHR was measured at 24 to 26 weeks' gestation. Outcome variables included survival and the need for extracorporeal membrane oxygenation (ECMO). RESULTS: Overall survival was 47%. LHR ranged from 0.62 to 1.86. No patient with an LHR of less than 1.0 (n = 3) survived despite ECMO, whereas all patients with an LHR greater than 1.4 survived (n = 4), one requiring ECMO. LHR values between 1.0 to 1.4 were associated with 38% survival (n = 8), 75% requiring ECMO. Overall, survivors had a mean LHR of 1.4 +/- 0.33 and nonsurvivors, 1.05 +/- 0.3 (P < .05). CONCLUSION: The LHR is a useful index to help predict neonatal outcome in patients who have left-sided CDH.

Slide tracheoplasty for congenital tracheal stenosis: a case report.

BACKGROUND/PURPOSE: A variety of techniques have been used to manage pediatric congenital tracheal stenosis. The authors report the technique of slide tracheoplasty for a child with long congenital tracheal stenosis. METHODS: A 2-year-old male presented with a history of stridor with feeding. Bronchoscopy findings showed 50% stenosis from complete cartilaginous rings, extending from 2.5 cm below the vocal cords to 2 cm above the carina. Through a neck incision, the trachea was exposed from the cricoid to both bronchi and transected at the midpoint of the stenosis. The upper trachea was split anteriorly to the area of stenosis just below the cricoid. The lower trachea was split posteriorly in the midline. Posterior dissection allowed sliding and anastomosis of both tracheal segments while the lateral vascular supply was left intact. A brace was placed to maintain cervical flexion, and the patient underwent extubation in the operating room. RESULTS: He recovered without complication and was dis charged on postoperative day 4. CONCLUSION: Slide tracheoplasty offers several advantages for tracheal reconstruction because it is performed with the native tracheal tissues, can be accomplished through a transverse collar incision, and can repair long stenoses without significant tracheal shortening.

Obesity and outcome following renal transplantation.

Single institution series have demonstrated that obese patients have higher rates of wound infection and delayed graft function (DGF), but similar rates of graft survival. We used UNOS data to determine whether obesity affects outcome following renal transplantation. From the UNOS database, we identified patients who underwent primary kidney-only transplantation between 1997 and 1999. Recipient and donor body mass index (BMI) was categorized as underweight (BMI < 18.5), normal (BMI 18.5-24.9), overweight (BMI 25-29.9), obese (BMI 30-34.9) or morbidly obese (BMI > or = 35). We correlated BMI with intermediate measures of graft outcome and overall graft survival, and created multivariate models to evaluate the independent effect of BMI on graft outcome, adjusting for factors known to affect graft success. The study sample comprised 27,377 recipients. Older age, female sex, African American race and increased comorbidity were associated with obesity (p < 0.001). Compared with normal weight patients, morbid obesity was independently associated with an increased risk of DGF (p < 0.001), prolonged hospitalization (p < 0.001), acute rejection (p = 0.006) and decreased overall graft survival (p = 0.001). Donor BMI did not affect overall graft survival (p > or = 0.07). Recipient obesity is associated with an increased risk of DGF and decreased graft survival following renal transplantation.

Adenovirus-mediated gene transfer in the midgestation fetal mouse.

BACKGROUND: The development of strategies for gene transfer in utero will make possible the amelioration, and eventually the cure, of genetic diseases associated with pre- and postnatal morbidity and mortality. We have developed a murine model for in utero, intrahepatic, adenovirus-mediated gene transfer in Day 15 fetuses and compared the level and distribution of luciferase reporter gene expression in newborns with those observed in adult animals injected intravenously. MATERIAL AND METHODS: CD-1 fetuses underwent intrahepatic injection on Day 15 of gestation with 1 x 10(7) particle-forming units (PFU) of an E1- and E3-deleted recombinant adenovirus containing the luciferase reporter gene or with normal saline. At birth, pups were euthanized, and the brain, heart, intestine, liver, lungs, and spleen harvested and analyzed for luciferase activity. RESULTS: Two adenovirus-injected litters proceeded to term and one female aborted. Tissues from 10 newborn mice in the experimental group and 5 newborns in the control group were analyzed; tissues from the remaining newborns were reserved for other studies. High-level luciferase expression was detected in all adenovirus-injected newborn livers. Lower levels of luciferase activity were detected in distant organs. Hepatic toxicity as determined by serum transaminase elevations was observed in adult, but not in newborn mice previously injected with the adeno-luciferase virus. CONCLUSIONS: In utero intrahepatic gene delivery with adenoviral vectors in the developing murine fetus is feasible and produces high-level gene expression. These studies suggest that viral and nonviral gene delivery vectors may be useful in the development of future approaches to prenatal treatment of genetic disorders.