Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally ...used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.
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He et al. present a robust strategy using imaging capillary isoelectric focusing to identify adeno-associated virus (AAV) vector viral capsid protein charged variants and present a corresponding study of deamidation impact on potency for this emerging gene therapy modality.
Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis ...can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.
The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney ...disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.
Physicochemical tests represent important tools for the analytical control strategy of biotherapeutics. For adenoviral modalities, anion-exchange high performance liquid chromatography (AEX-HPLC) ...represents an important methodology, as it is able to simultaneously provide information on viral particle concentration, product purity and surface charge in a high-throughput manner. During product development of an adenoviral-based therapeutic, an accelerated stability study was performed and showed changes in each of the AEX-HPLC reportable attributes. These changes also correlated with a decrease in product infectivity prompting a detailed characterization of the impurity and mechanism of the surface charge change. Characterization experiments identified the impurity to be free hexon trimer, suggesting that capsid degradation could be contributing to both the impurity and reduced particle concentration. Additional mass spectrometry characterization identified deamidation of specific hexon residues to be associated with the external surface charge modification observed upon thermal stress conditions. To demonstrate a causal relationship between deamidation and surface charge changes observed by AEX-HPLC, site-directed mutagenesis experiments were performed. Through this effort, it was concluded that deamidation of asparagine 414 was responsible for the surface charge alteration observed in the AEX-HPLC profile but was not associated with the reduction in infectivity. Overall, this manuscript details critical characterization efforts conducted to enable understanding of a pivotal physicochemical test for adenoviral based therapeutics.
Purpose
Glenoid loosening is a common failure mechanism of anatomic total shoulder arthroplasty (TSA). New hybrid fixation glenoids allow bony ingrowth and decrease the rates of glenoid loosening. ...The purpose of this study is to describe a new failure mode of polyethylene dissociation from the ingrowth cage in a single implant design.
Methods
A retrospective review was performed using an institutional shoulder arthroplasty database of a single hybrid cage glenoid (Exactech, Gainesville, FL). Implants demonstrating this failure mechanism were investigated.
Results
Five out of 206 (2.4%) primary TSAs with cage glenoids performed at our institution and 2 referred for revision suffered glenoid articular face failure. Mean age was 57 (range 38–67 years). Two of 7 failures (29%) occurred secondary to trauma. Failure occurred at mean 14 months after index arthroplasty (range 0–30 months). Revision occurred at mean 24 months after index arthroplasty (range 6–39 months). Six of the 7 patients (86%) had posteriorly augmented glenoids. All 6 patients who had available pre-failure radiographs demonstrated off-axis deviation between the peripheral pegs and central cage (mean 4.8°, range 3°–6°), which may predispose the implant to failure by pre-stressing the material interface.
Conclusion
Failure between the glenoid articular face and the central ingrowth cage is a unique failure mechanism to modular hybrid ingrowth glenoids. We hypothesize that this is predisposed by off-axis drilling leading to pre-stressing of the material interface. Surgeons should be aware of the existence of this failure mechanism, particularly when seeing patients with acute pain after an otherwise unproblematic TSA.
Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the ...experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 μL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.
We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this ...outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.
A central goal of The Academy of Breastfeeding Medicine is the development of clinical protocols for managing common medical problems that may impact breastfeeding success. These protocols serve only ...as guidelines for the care of breastfeeding mothers and infants and do not delineate an exclusive course of treatment or serve as standards of medical care. Variations in treatment may be appropriate according to the needs of an individual patient.
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