Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 µg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV.

Vector genome loss and epigenetic modifications mediate decline in transgene expression of AAV5 vectors produced in mammalian and insect cells.

Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (∼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.

Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype 5 (AAV5)-based gene therapy vector containing a B-domain-deleted human coagulation factor VIII (hFVIII) gene controlled by a liver-selective promoter. AAV5-hFVIII-SQ is currently under clinical investigation as a treatment for severe hemophilia A. The full-length AAV5-hFVIII-SQ is >4.9 kb, which is over the optimal packaging limit of AAV5. Following administration, the vector must undergo a number of genome-processing, assembly, and repair steps to form full-length circularized episomes that mediate long-term FVIII expression in target tissues. To understand the processing kinetics of the oversized AAV5-hFVIII-SQ vector genome into circular episomes, we characterized the various molecular forms of the AAV5-hFVIII-SQ genome at multiple time points up to 6 months postdose in the liver of murine and non-human primate models. Full-length circular episomes were detected in liver tissue beginning 1 week postdosing. Over 6 months, quantities of circular episomes (in a predominantly head-to-tail configuration) increased, while DNA species lacking inverted terminal repeats were preferentially degraded. Levels of duplex, circular, full-length genomes significantly correlated with levels of hFVIII-SQ RNA transcripts in mice and non-human primates dosed with AAV5-hFVIII-SQ. Altogether, we show that formation of full-length circular episomes in the liver following AAV5-hFVIII-SQ transduction was associated with long-term FVIII expression.

Development of a functional bioassay for arylsulfatase B using the natural substrates of the enzyme.

A functional bioassay has been developed for measuring the intracellular activity of recombinant human arylsulfatase B (rhASB) on its natural glycosaminoglycan (GAG) substrates, dermatan sulfate (DS), and chondroitin sulfate (CS) when the enzyme is taken up into cultured ASB-deficient human fibroblasts (GM00519). The enzyme ASB is a lysosomal exohydrolase, cleaving sulfate from the N-acetylgalactosamine-4-sulfate (GalNAc-4S) residue at the nonreducing terminal of GAG structures. ASB-deficient cells accumulate DS and CS, which may be partially hydrolyzed by other lysosomal hydrolases, with the reactions stopping if a GalNAc-4S residue is reached on the nonreducing end of the oligosaccharide. When rhASB is added to the culture medium, the enzyme is taken up and translocates to the lysosomes and the intracellular DS and CS are depleted, demonstrating that the uptake of rhASB is able to restore lysosomal function in an in vitro cell-based assay. The accumulation and depletion of DS and CS are measured by digesting the residual intracellular DS and CS content with chondroitin ABC lyase and monitoring a characteristic disaccharide digestion product by laser-induced fluorescence-capillary zone electrophoresis (LIF-CZE). In the proposed assay format, GM00519 cells are cultured 5 weeks postconfluence to accumulate DS/CS, followed by incubation with rhASB (1-20 pM) for 5 days, and the CS/DS depletion profiles are compared between samples. The assay measures depletion of DS/CS independently of their molecular size or processing state; in this approach, all DS- and CS-like substances accumulating in the absence of ASB activity are considered to be natural substrates of the enzyme.

Development of parallel line analysis criteria for recombinant adenovirus potency assay and definition of a unit of potency.

Parameter settings of a parallel line analysis procedure were defined by applying statistical analysis procedures to the absorbance data from a cell-based potency bioassay for a recombinant adenovirus, Adenovirus 5 Fibroblast Growth Factor-4 (Ad5FGF-4). The parallel line analysis was performed with a commercially available software, PLA 1.2. The software performs Dixon outlier test on replicates of the absorbance data, performs linear regression analysis to define linear region of the absorbance data, and tests parallelism between the linear regions of standard and sample. Width of Fiducial limit, expressed as a percent of the measured potency, was developed as a criterion for rejection of the assay data and to significantly improve the reliability of the assay results. With the linear range-finding criteria of the software set to a minimum of 5 consecutive dilutions and best statistical outcome, and in combination with the Fiducial limit width acceptance criterion of <135%, 13% of the assay results were rejected. With these criteria applied, the assay was found to be linear over the range of 0.25 to 4 relative potency units, defined as the potency of the sample normalized to the potency of Ad5FGF-4 standard containing 6 x 10(6) adenovirus particles/mL. The overall precision of the assay was estimated to be 52%. Without the application of Fiducial limit width criterion, the assay results were not linear over the range, and an overall precision of 76% was calculated from the data. An absolute unit of potency for the assay was defined by using the parallel line analysis procedure as the amount of Ad5FGF-4 that results in an absorbance value that is 121% of the average absorbance readings of the wells containing cells not infected with the adenovirus.

Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs.

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE-19) using three different lots of fetal bovine serum (FBS-Ia, FBS-Ib, and FBS-II). We found that the growth rate of the culture was significantly and consistently higher in the FBS-II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional-related and cell-spreading-related proteins, decreased over time.

A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.

An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6. This E1-transformed human cell line contains only Ad5 nucleotides 459-3510, which precludes double crossover-type homologous recombination because the Ad5FGF-4 only contains 5' Ad5 sequence up to nucleotide 453. The Ad5FGF4 vector does, however, retain 177 nucleotides of the 3' end of the E1B-55K gene that are also present in PER.C6. With only this single region of homology between vector and cell line, we were surprised to detect virus-specific cytopathic effects (CPE) in our cell-based assay for RCA. This CPE-inducing agent was amplified in nonpermissive A549 cells but also supported amplification of the parental Ad5FGF-4. Because we were unable to isolate the CPE-inducing agent in pure form we first identified it as atypical RCA. Polymerase chain reaction (PCR) and Southern blot experiments identified viral DNA segments in which recombination had occurred between the 177 nucleotides of E1B present in both Ad5FGF-4 and PER.C6. The atypical RCA genomes contain a copy of the original (PGK promoter-E1 gene carrying) plasmid used in the construction of the PER.C6 cell line and they retained the parental FGF-4 transgene. However, significant deletions occurred within the recombined genomes in compensation for the large insertion from PER.C6 sequences and resulted in the loss of essential viral genes. This deletion renders these recombinant viruses replication defective, requiring helper functions from remaining parental Ad5FGF-4 for amplification. These atypical RCA entities may be more properly designated as helper-dependent E1-positive particles (HDEPs). This finding shows the importance of avoiding the use of "nonmatched" vectors where any overlap exists between the recombinant vector and E1 sequences in the packaging cell line. The cloning of the FGF-4 transgene into an adenoviral vector specifically "matched" for PER.C6 (lacking the 177 nucleotide region of homology) has allowed extensive virus propagation (Ad5.1FGF-4) with no CPE- or HDEP-like events yet detected.

Quantification of bifunctional diethylenetriaminepentaacetic acid derivative conjugation to monoclonal antibodies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The results of the characterization of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based method that was developed to establish the stoichiometry of CHX-A''-diethylenetriaminepentaacetic acid (DTPA) or benzyl-DTPA conjugated to a recombinant immunoglobulin G (IgG) are reported. This simple method does not require an accurate measurement of the sample protein concentration to accurately quantify the number of DTPA conjugated. It is also not necessary to thoroughly remove nonconjugated DTPA from the sample. The average number of moles of DTPA attached per mole of IgG was calculated from the difference in the observed masses of DTPA-IgG and nonconjugated IgG divided by the molecular weight of the DTPA derivative. As more DTPA is attached, the [M+H](+) peak of DTPA-IgG becomes broader and noisier. Also, the signal intensity in the mass spectrum decreases, apparently due to the increase in the heterogeneity in the number of DTPA attached to each molecule of IgG. The standard deviation of the measured mass and that of the stoichiometry of the DTPA attached per IgG increased as more DTPA was attached. The standard deviation, expressed as coefficient of variation for samples with 2 to 4 mol of DTPA attached per mole of IgG, was 8 to 9%.

Precise and comparative pegylation analysis by microfluidics and mass spectrometry.

Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.

A potency assay for a replication incompetent adenovirus type 5 carrying a human fgf-4 gene.

A bioassay that measures the potency of the FGF-4 transgene carried by a replication incompetent adenovirus type 5, Ad5FGF-4, was developed on ARPE-19 cells. The assay is carried out in a microtiter plate format and measures cellular proliferation following infection of ARPE-19 cells with a serial dilution of Ad5FGF-4. Proliferation is measured as a percentage increase in absorbance reading in relation to a mock-infected control. Ad5LacZ and Ad5FGF-4 viruses treated similarly to the test sample are included as negative and positive controls, respectively. The increased absorbance reading resulting from infection with the virus correlates with FGF-4 production as determined by an FGF-4 enzyme-linked immunosorbent assay, an increase in de novo DNA synthesis as measured by BrdU incorporation, and an increase in the total cell number. The assay shows a dose-dependent response and is capable of evaluating the stability of Ad5FGF-4. A sample being tested is compared with a reference standard, and the relative potency value is obtained by a parallel line analysis of the dose-response curve of the test article in relation to the reference standard. Therefore, this procedure can be used as an in vitro efficacy-indicating assay, demonstrating that the FGF-4 transgene product carried by Ad5FGF-4 is biologically active.

Electrochemical Detectors in HPLC and Ion Chromatography.

Back in 1952, the renowned Polish electrochemist Wiktor Kemula introduced chromato-polarography,1 i.e., polaro-graphic detection for liquid chromatography. This technique continued to develop slowly until the early 1970s (for a review see Reference 2) when modem high-performance liquid chromatography (HPLC) emerged. This new, highly efficient chromatographc method could only be. used with detectors ensuring low dispersion. It was not easy to modify the dropping mercury electrode cells to satisfy this requirement. However, at the same time, electroanalytical chemists, who already had much experience in using carbon-based electrodes for oxidative detection in flow analysis, put forward the idea of oxidative amperometric detection in liquid chromatography.3,4 In this technique, solid or quasi-solid (paste) electrodes were used and this made possible the construction of miniaturized cells with just a few microliter volume.

Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches.

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods.

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.