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.

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.

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.

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.

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.

In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

Reexpression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration.

Adenovirus-mediated gene delivery is limited by the induction of immune responses that produce toxicity and prevent reexpression. To determine whether adenoviral delivery in the preimmune fetus would produce tolerance, we assessed luciferase (luc) expression following sequential pre- and postnatal adenoviral-mediated gene delivery. Day 15 fetuses were injected intrahepatically with 1 x 10(7) pfu of an adenoviral-luc vector (Ad-luc). Following in utero injection, hepatic luc expression persisted 1 month postnatally. No humoral response to adenovirus or luc was detected. Adult mice, previously injected in utero, were reinjected intravenously with 5 x 10(8) pfu of Ad-luc at 3 months of age and again at 6 months with either 5 x 10(8) pfu of Ad-luc or cationic liposome-DNA complexes (CLDC). Following the first postnatal injection, animals injected in utero had levels of luc comparable to those of age-matched naive controls. However, both control and experimental animals subsequently developed antibodies to adenovirus and luc. No further expression was achieved with a second postnatal injection of Ad-luc or with delivery of CLDC-luc. These studies demonstrate that the delivery of adenoviral vectors in utero at E15 does not elicit an immune response. However, delivery of recombinant adenovirus postnatally results in brisk and limiting immune responses regardless of the in utero exposure.

Cord ultrasonic transection procedure for selective termination of a monochorionic twin.

Placental vascular communications can present a life-threatening problem in monochorionic twins when one fetus has a lethal anomaly. Although selective feticide is the best option for salvaging the normal twin, techniques normally employed (i.e. intracardiac potassium, air embolism) are not prudent given the common circulatory system. Furthermore, in monoamniotic, monochorionic twin gestations it is important to transect the umbilical cord completely to prevent entanglement of the dead fetus around the cord of the normal twin. We present two cases of monochorionic twins in which the cords were transected with a harmonic scalpel under ultrasonic guidance via one trocar. The harmonic scalpel is an instrument which can simultaneously coagulate and cut blood vessels or tissues. The cord ultrasonic transection procedure is a novel, minimally invasive technique which offers several advantages over the methods currently used for selective feticide in discordant monochorionic twin gestations.

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.

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.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero.

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (X(H)X), and FVIII-deficient (X(H)Y and X(H)X(H)) fetuses injected with adenoviral vectors carrying luciferase or beta-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 x 10(5) plaque-forming units) was 87%. FVIII activity in plasma was 50.7 +/- 10.5% of normal levels at day 2 of life, 7.2 +/- 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

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.

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.

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.

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.

Are bilateral fetal lung masses double trouble?

OBJECTIVES: To examine the perinatal natural history of bilateral fetal cystic lung masses. METHODS: The records of a tertiary medical center over a 3-year period were reviewed for cases of fetal bilateral pulmonary masses. RESULTS: Three of 98 fetuses referred for evaluation over a 3-year period from September of 1995 to August of 1998 had bilateral lung lesions. Two of these cases resulted in live births, while one, associated with hydrops, resulted in death after preterm delivery. CONCLUSIONS: Similar to prenatally diagnosed unilateral lung lesions, hydropic fetuses with bilateral cystic lung lesions have a poor prognosis. Nonhydropic fetuses, however, may be asymptomatic despite persistent lesions on postnatal CT scan. These observation may prove helpful for prenatal counseling for these rare lesions.