Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection

Chain Deformation for a Polymer Melt under Shear

We used a Couette cell placed within an NMR microscopy apparatus to examine molecular alignment in polydimethylsiloxane melts under shear. 1H dipolar interactions are used to examine main chain ordering while 2H NMR quadrupole splittings are measured in a deuterated oligomer probe, in each case selectively observing different alignment orientations with respect to the flow field. While the main chain results are consistent with the alignment tensor as measured by birefringence, the oligomer senses a uniaxial environment whose director is normal to the velocity-vorticity plane. In the light of these results we propose supramolecular ordering involving polymer stratification

Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction

Arsenic Trioxide Stabilizes Accumulations of Adeno-Associated Virus Virions at the Perinuclear Region, Increasing Transduction In Vitro and In Vivo

Interactions with cellular stress pathways are central to the life cycle of many latent viruses. Here, we utilize adeno-associated virus (AAV) as a model to study these interactions, as previous studies have demonstrated that cellular stressors frequently increase transduction of recombinant AAV (rAAV) vectors and may even substitute for helper virus functions. Since several chemotherapeutic drugs are known to increase rAAV transduction, we investigated the effect of arsenic trioxide (As2O3), an FDA-approved chemotherapeutic agent with known effects on several other virus life cycles, on the transduction of rAAV. In vitro, As2O3 caused a dose-dependent increase in rAAV2 transduction over a broad range of cell lines from various cell types and species (e.g., HEK-293, HeLa, HFF hTERT, C-12, and Cos-1). Mechanistically, As2O3 treatment acted to prevent loss of virions from the perinuclear region, which correlated with increased cellular vector genome retention, and was distinguishable from proteasome inhibition. To extend our investigation of the cellular mechanism, we inhibited reactive oxygen species formation and determined that the As2O3-mediated increase in rAAV2 transduction was dependent upon production of reactive oxygen species. To further validate our in vitro data, we tested the effect of As2O3 on rAAV transduction in vivo and determined that treatment initiated transgene expression as early as 2 days posttransduction and increased reporter expression by up to 10-fold. Moreover, the transduction of several other serotypes of rAAV was also enhanced in vivo, suggesting that As2O3 affects a pathway used by several AAV serotypes. In summary, our data support a model wherein As2O3 increases rAAV transduction both in vitro and in vivo and maintains perinuclear accumulations of capsids, facilitating productive nuclear trafficking

Prediction of adeno-associated virus neutralizing antibody activity for clinical application

Patients with neutralizing antibodies (Nab) against adeno-associated virus (AAV) are usually excluded from treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including: sera volume, AAV dose/cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a 4-fold increase in sensitivity for measurement of Nab titers was observed compared to an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various Phase I clinical trials

Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair

Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders

Probing molecular ordering in the nematic phases of para-linked bimesogen dimers through NMR studies of flexible prochiral solutes

The quadrupolar splittings of perdeuteriated n-decane dissolved in nematic phases formed by mesogenic dimers of the CBnCB series, for n = 7,9,10,11, are measured throughout the entire temperature range of these phases. These results are reported together with related measurements using the common nematic phase of 5CB as a solvent for n-decane. The data obtained from the 13C spectra of the cyanobiphenyl mesogenic units of the monomeric and dimeric solvent molecules yield the order parameter of those units. The information obtained from this set of experiments is used to elucidate the structure of the low temperature (NX) and the high temperature (N) nematic phases of CBnCB dimers with n = 7,9,11. The polar twisted nematic (NPT) model is found to provide a consistent description not only of our experimental results but also of NMR measurements previously reported in the literature for these phases. These findings suggest that the high temperature nematic (N) is not a common, locally uniaxial and apolar nematic, but rather a nematic phase consisting of NPT clusters. The twist-bend (NTB) model, often identified with the NX phase, is shown to be inadequate to account even qualitatively for crucial features of the experimental findings

2,3 and 2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection