Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia.

Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector and has recently been used in patients with hemophilia. One limitation of AAV application is that most humans have experienced wild-type AAV serotype 2 exposure, which frequently generates neutralizing antibodies (NAbs) that may inhibit rAAV2 vector transduction. Employing alternative serotypes of rAAV vectors may circumvent this problem. We investigated the development of NAbs in early childhood by examining sera gathered prospectively from 62 children with hemophilia A, participating in a multi-institutional hemophilia clinical trial (the Joint Outcome Study). Clinical applications in hemophilia therapy have been suggested for serotypes AAV2, AAV5 and AAV8, therefore NAbs against these serotypes were serially assayed over a median follow-up of 4 years. NAbs prevalence increased during early childhood for all serotypes. NAbs against AAV2 (43.5%) were observed more frequently and at higher titers compared with both AAV5 (25.8%) and AAV8 (22.6%). NAbs against AAV5 or AAV8 were rarely observed in the absence of co-prevalent and higher titer AAV2 NAbs, suggesting that NAbs to AAV5 and AAV8 were detected following AAV2 exposure due to partial cross-reactivity of AAV2-directed NAbs. The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs.

Debridement, antibiotics, and implant retention in non-oncological femoral megaprosthesis infections: minimum 5 year follow-up.

PURPOSE: Megaprostheses are increasingly utilised outside of the oncological setting, and remain at significant risk of periprosthetic joint infection (PJI). Debridement, antibiotic, and implant retention (DAIR) is an established treatment for PJI, however its use in non-oncological patients with femoral megaprostheses has not been widely reported. There are significant differences in patient physiology, treatment goals, and associated risks between these patient cohorts. METHODS: We identified 14 patients who underwent DAIR for a PJI of their femoral megaprostheses, between 2000 and 2014, whom had their index procedure secondary to non-oncological indications. Patients were managed as part of a multidisciplinary team, with our standardised surgical technique including exchange of all mobile parts, and subsequent antibiotic therapy for a minimum of 3 months. Patients were followed up for a minimum of 5 years. RESULTS: Patients included six proximal femoral replacements, five distal femoral replacements, and three total femoral replacements. No patients were lost to follow-up. There were six males and eight females, with a mean age of 67.2 years, and mean ASA of 2.3. Nine patients (64.3%) successfully cleared their infection following DAIR at a minimum of 5 year follow-up. Five patients (35.7%) required further revision surgery, with four patients cleared of infection. No patients who underwent DAIR alone suffered complications as a result of the procedure. CONCLUSIONS: The use of DAIR in these complex patients can lead to successful outcomes, but the risk of further revision remains high. The success rate (64.3%) remains on par with other studies evaluating DAIR in megaprostheses and in primary arthroplasty. This study indicates judicious use of DAIR can be an appropriate part of the treatment algorithm. LEVEL OF EVIDENCE: II.

Peptide affinity reagents for AAV capsid recognition and purification.

We report the discovery of AAV capsid-binding peptides identified through phage panning. The heptapeptide motif GYVSRHP selectively recognized AAV serotype 8 capsids and blocked transduction in vitro. Recombinant AAV8 vectors were purified directly from crude cell lysate and supernatant through sequential application of peptide affinity and anion exchange chromatography. Peptide affinity reagents may serve as useful alternatives to monoclonal antibodies in AAV capsid recognition, and offer readily scalable solutions for purification of clinical grade AAV vectors.

A small regulatory element from chromosome 19 enhances liver-specific gene expression.

Tissue-specific promoters for gene therapy are typically too big for adeno-associated virus (AAV) vectors; thus, the exploration of small effective non-viral regulatory elements is of particular interest. Wild-type AAV can specifically integrate into a region on human chromosome 19 termed AAVS1. Earlier work has determined that a 347 bp fragment (Chr19) of AAVS1 has promoter and transcriptional enhancer activities. In this study, we further characterized this genetic regulation and investigated its application to AAV gene therapy in vitro and in vivo. The Chr19 347 bp fragment was dissected into three regulatory elements in human embryonic kidney cells: (i) TATA-independent promoter activity distributed throughout the fragment regardless of orientation, (ii) an orientation-dependent insulator function near the 5' end and (iii) a 107 bp enhancer region near the 3' end. The small enhancer region, coupled to the mini-CMV promoter, was used to drive the expression of several reporters following transduction by AAV2. In vivo data demonstrated enhanced transgene expression from the Chr19-mini-CMV promoter cassette after tail vein injection primarily in the liver at levels comparable to the chicken beta-actin promoter and higher than the liver-specific TTR promoter (>2-fold). However, we did not observe this increase after muscle injection, suggesting tissue-specific enhancement. All of the results support identification of a small DNA fragment (347 bp) from AAV Chr19 integration site capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors.

Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals.

Current technologies for visualizing infectious pathways of viruses rely on fluorescent labeling of capsid proteins by chemical conjugation or genetic manipulation. For noninvasive in vivo imaging of such agents in mammalian tissue, we engineered bioluminescent Gaussia luciferase-tagged Adeno-associated viral (gLuc/AAV) vectors. The enzyme was incorporated into recombinant AAV serotypes 1, 2 and 8 capsids by fusing to the N-terminus of the VP2 capsid subunit to yield bioluminescent virion shells. The gLuc/AAV vectors were used to quantify kinetics of cell-surface-binding by AAV2 capsids in vitro. Bioluminescent virion shells displayed an exponential decrease in luminescent signal following cellular uptake in vitro. A similar trend was observed following intramuscular injection in vivo, although the rate of decline in bioluminescent signal varied markedly between AAV serotypes. gLuc/AAV1 and gLuc/AAV8 vectors displayed rapid decrease in bioluminescent signal to background levels within 30 min, whereas the signal from gLuc/AAV2 vectors persisted for over 2 h. Bioluminescent virion shells might be particularly useful in quantifying dynamics of viral vector uptake in cells and peripheral tissues in live animals.

Renal prostaglandins, effective renal plasma flow and glomerular filtration rate in healthy elderly subjects.

This study tests the hypothesis that the age-associated reduction in glomerular filtration rate (GFR) and the presence of glomerulosclerosis renders effective renal plasma flow (ERPF) prostaglandin dependent. Ten healthy elderly volunteers were studied in a single-blind placebo-controlled manner using indomethacin to suppress the renal prostaglandins. There was no significant difference in ERPF or GFR following indomethacin when compared with placebo. These results suggest that blocking renal prostaglandins does not significantly alter ERPF or GFR in healthy elderly people.

Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.

Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.

Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form.

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.