Validity Evidence From Ratings of Pediatric Interns and Subinterns on a Subset of Pediatric Milestones.

PURPOSE: To investigate evidence for validity of faculty members' pediatric milestone (PM) ratings of interns (first-year residents) and subinterns (fourth-year medical students) on nine subcompetencies related to readiness to serve as a pediatric intern in the inpatient setting. METHOD: The Association of Pediatric Program Directors Longitudinal Educational Assessment Research Network (APPD LEARN) and the National Board of Medical Examiners collaborated to investigate the utility of assessments of the PMs for trainees' performance. Data from 32 subinterns and 179 interns at 17 programs were collected from July 2012 through April 2013. Observers used several tools to assess learners. At each site, a faculty member used these data to make judgments about the learner's current developmental milestone in each subcompetency. Linear mixed models were fitted to milestone judgments to examine their relationship with learner's rank and subcompetency. RESULTS: On a 5-point developmental scale, mean milestone levels for interns ranged from 3.20 (for the subcompetency Work effectively as a member of a team) to 3.72 (Humanism) and for subinterns from 2.89 (Organize and prioritize care) to 3.61 (Professionalization). Mean milestone ratings were significantly higher for the Professionalism competency (3.59-3.72) for all trainees compared with Patient Care (2.89-3.24) and Personal and Professional Development (3.33-3.51). Mean intern ratings were significantly higher than mean subintern ratings for all nine subcompetencies except Professionalization, Humanism, and Trustworthiness. CONCLUSIONS: The PMs had a coherent internal structure and could distinguish between differing levels of trainees, which supports their validation for documenting developmental progression of pediatric trainees.

Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration.

The safety and efficacy of gene therapy for inherited retinal diseases is being tested in humans affected with Leber's congenital amaurosis (LCA), an autosomal recessive blinding disease. Three independent studies have provided evidence that the subretinal administration of adeno-associated viral (AAV) vectors encoding RPE65 in patients affected with LCA2 due to mutations in the RPE65 gene, is safe and, in some cases, results in efficacy. We evaluated the long-term safety and efficacy (global effects on retinal/visual function) resulting from subretinal administration of AAV2-hRPE65v2. Both the safety and the efficacy noted at early timepoints persist through at least 1.5 years after injection in the three LCA2 patients enrolled in the low dose cohort of our trial. A transient rise in neutralizing antibodies to AAV capsid was observed but there was no humoral response to RPE65 protein. The persistence of functional amelioration suggests that AAV-mediated gene transfer to the human retina does not elicit immunological responses which cause significant loss of transduced cells. The persistence of physiologic effect supports the possibility that gene therapy may influence LCA2 disease progression. The safety of the intervention and the stability of the improvement in visual and retinal function in these subjects support the use of AAV-mediated gene augmentation therapy for treatment of inherited retinal diseases.

Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.

BACKGROUND: Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. METHODS: We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS: AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION: The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING: Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Safety and efficacy of gene transfer for Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose(xl) are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 x 10(14)vg and 2.52 x 10(15) empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 x 10(14)vg containing 0.25 x 10(14) empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.

Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5.

Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.

Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization.

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10- to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.