The role of membrane chemistry in Lentiviral vector clarification recovery for cell and gene therapies

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Automated filtration screening of lentiviral vectors with multiple envelope proteins

Lentiviral Vectors (LV) have been shown to successfully transfer therapeutic genes into dividing and non-dividing cells in laboratory and clinical environments for the benefit of cell and gene therapies. Current LV production features an initial clarification stage to remove cellular debris in addition to viral and serum protein aggregates prior to further downstream processing. Such filtration tasks have illustrated decreases in titer of vectors potentially via damage to external envelope proteins or the unwanted retention of particles [1]. LV production is generally characterized by its fragility and careful downstream processing design is required to ensure high recovery and purity of vectors. Evidence suggests that the selection of salt concentration and pH affects the aggregation propensity of proteins and the binding of vectors and contaminants to filters such as that seen with adeno-associated virus processing [2] whilst also negatively impacting the infectivity of the vector [3]. Such conditions need to be evaluated to ensure effective processing if vector development is to proceed to meet future demands. A design of experiment definitive scree model was implemented in a Tecan liquid handling platform to rapidly screen various filters under different salt concentrations and pH ranges. Vectors containing the viral envelope proteins VSV-G, Cocal-G and RDPro was filtered across four membrane filter types. The vector transmission was measured by reverse transcriptase activity as a % of unfiltered product, and total protein transmission by Bradford assay. Data has shown vector and total protein transmission is not strongly affected by salt concentration, whereas pH 9 shows improved LV transmission across all envelopes and filters tested. RDPro enveloped LV report strongest filter transmission, whilst Cocal-G and VSV-G remain similar in efficiency. The highest reported LV transmission was found in filters with PVDF chemistry, whereas the best performer for protein removal was PES microwells. Positive correlation between LV and protein transmission was also seen. The work increases our understanding of how filtration affects initial clarification of vectors of differing envelope proteins harvested from cell culture and attempts to characterize the impact of salt concentration and pH value. In identifying the impact of such conditions on vectors, work can continue to improve LV processing, leading to ideal and scalable solutions to address demand for vectors in cell and gene therapies. [1] Merten O, Charrier S, Laroudie N, Fauchille S, Dugué C, Jenny C et al. Large-Scale Manufacture and Characterization of a Lentiviral Vector Produced for Clinical Ex Vivo Gene Therapy Application. Human Gene Therapy. 2011;22(3):343-356. [2] Wright J, Le T, Prado J, Bahr-Davidson J, Smith P, Zhen Z et al. Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation. Molecular Therapy. 2005;12(1):171-178. [3] de las Mercedes Segura M, Kamen A, Trudel P, Garnier A. A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography. Biotechnology and Bioengineering. 2005;90(4):391-4

Insights into product and process related challenges of lentiviral vector bioprocessing

Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale-down technologies. The impact of shear during membrane filtration in a tangential flow filtration-mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems

Superinfection arising in stable lentiviral vector producer cell lines bearing Cocal-G envelope proteins

Lentiviral vectors (LV) have been shown to successfully transfer therapeutic genes into dividing and non-dividing cells in laboratory and clinical environments for the benefits of cell and gene therapies. Current LV production chiefly relies on a transient transfection method, wherein HEK 293T cells are transfected with 3-4 plasmids. Such methods have shown batch to batch variability, and increased costs due to the requirements of considerable quantities of cGMP plasmids at clinical stages [1]. This can be circumvented using stable producer cell lines, such as the WinPac cell line, that stably harbor all constructs required for vector production and reliably output LV vectors over long periods of time [2]. However, the commonly pseudotyped LV envelope protein, VSV-G, has difficulty in long term expression and is inactivated by complement [3] and therefore alternatives must be sought. Such alternatives can be found in the Cocal-G envelope protein, which can be expressed long term, is resistant to complement, and bears similarity to VSV-G whereby both derive within the same vesiculovirus genus [4]. A stable LV producer using Cocal-G envelope in the WinPac cell line was produced. Results have illustrated that Cocal-G envelope protein expression leads to superinfection of the LV producing cell line, creating long term instability due to accumulation of the GFP transgene as determined by qPCR. Such superinfection can be prevented by the addition of the non-nucleoside reverse transcriptase inhibitor nevirapine to the cell culture media, leading to protection from superinfection in long term culture. The antiviral can subsequently be removed by buffer exchange in Vivaspin 4 ultrafiltration cassettes (100,000 MWCO), regenerating infectious titre of LV and suggests antiviral addition in upstream production does not negatively impact downstream purification. The cocal enveloped producer cell line was therefore robust enough to be scaled up for large scale LV harvesting as indicated from scaling to a Corning HYPERFlask system. This work increases our understanding of how LV envelope design may impact superinfection and ultimately specific productivity once cell progress further down a development pathway. In identifying the importance of envelope choice and necessary precautions as a result, work can continue to improve stable LV producers, leading to scalable solutions to address demand for vectors in cell and gene therapies. [1] Cornetta K, Reeves L, Cross S. Production of Retroviral Vectors for Clinical Use. Methods in Molecular Biology. 2008;:17-32. [2] Sanber K, Knight S, Stephen S, Bailey R, Escors D, Minshull J et al. Construction of stable packaging cell lines for clinical lentiviral vector production. Scientific Reports. 2015;5(1). [3] DePolo N, Reed J, Sheridan P, Townsend K, Sauter S, Jolly D et al. VSV-G Pseudotyped Lentiviral Vector Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy. 2000;2(3):218-222. [4] Humbert O, Gisch D, Wohlfahrt M, Adams A, Greenberg P, Schmitt T et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy. 2016;24(7):1237-1246

Nanofiber based lentiviral vector production

Viral vectors are an indispensable part of gene therapy clinical trials and lentiviral vectors (LVs) are becoming significant tools in the field. Unlike other retroviral vectors, they can transduce non-dividing cells thus providing for a wider range of potential applications. Current cultivation methods produce titers of 105 to 107 TU/mL of cell culture supernatant, which is not convenient for clinical trial requirements of 1011-1012 TU per patient [1], [2]. Therefore, it is necessary to concentrate the LV preparations and to remove process related impurities (e.g. serum proteins) and product related impurities, importantly including non-infective virus, as they can cause unwanted inflammation in patients. Small-scale purification can be achieved by ultracentrifugation but there are several disadvantages to this approach: the method is time consuming, there are limited scale-up possibilities, some impurities can be co-purified, and the success of the process is strongly dependent on well trained operator’s skills. Alternative methods that can provide for scalable production include tangential flow filtration (TFF) and chromatography. Currently, chromatography is dominated by porous bead stationary phases, which are optimized for purification of small proteins such as mAbs. This is not adequate for LV purification since binding sites located within particle pores are typically not accessible to macromolecular complexes such as viral vectors therefore alternative stationary phases are necessary. One such material is Puridify\u27s FibroSelect cellulose nanofibers. Due to its structural properties, this new purification platform provides high surface area and high capacity for viral vectors. High working flow rates are also possible due to excellent mass transfer properties based on convection, not diffusion that is typically seen in bead-based resins. [3]. In order to circumvent problems associated with transient plasmid transfection and the consequent removal of the plasmid material, we used a continuous producer cell line WinPac-RD [4] and HYPERFlask system for production of LV material. This vector has an RD-pro envelope protein and GFP reporter gene. The recovery through the purification process was monitored by several different methods: infectivity assay utilized GFP expression determined by flow cytometry, LV RNA genome was quantified via RT-qPCR using primers specific for GFP gene, LV particles were detected with p24 ELISA and SYBR Green I-based product-enhanced reverse transcriptase (SG-PERT) assays. By using TFF we were able to remove more than 99% of cell culture proteins, but LV recovery was less than 20%. While losses caused by diafiltration could be mitigated by adding stabilizing agents to the diafiltration buffer, the biggest loss occurred in the concentration step and the overall infectivity recovery remained low. This led us to investigate the implementation of a TFF-free nanofiber step based on ion-exchange chromatography to concentrate LV and eliminate a significant amount of impurities while maintaining high yield of a functional vector. [1] M. M. Segura, M. Mangion, B. Gaillet, and A. Garnier, “New developments in lentiviral vector design, production and purification.,” Expert Opin. Biol. Ther., vol. 13, no. November, pp. 987–1011, 2013. [2] R. R. MacGregor, “Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects,” Hum. Gene Ther., vol. 12, no. 16, pp. 2028–2029, 2001. [3] O. Hardick, S. Dods, B. Stevens, and D. G. Bracewell, “Nanofiber adsorbents for high productivity continuous downstream processing,” J. Biotechnol., vol. 213, pp. 74–82, 2015. [4] K. S. Sanber, S. B. Knight, S. L. Stephen, R. Bailey, D. Escors, J. Minshull, G. Santilli, A. J. Thrasher, M. K. Collins, and Y. Takeuchi, “Construction of stable packaging cell lines for clinical lentiviral vector production.,” Sci. Rep., vol. 5, p. 9021, 2015

Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo

editorial reviewedSynthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

peer reviewedIn addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke

Shrinking a large dataset to identify variables associated with increased risk of Plasmodium falciparum infection in Western Kenya

Large datasets are often not amenable to analysis using traditional single-step approaches. Here, our general objective was to apply imputation techniques, principal component analysis (PCA), elastic net and generalized linear models to a large dataset in a systematic approach to extract the most meaningful predictors for a health outcome. We extracted predictors for Plasmodium falciparum infection, from a large covariate dataset while facing limited numbers of observations, using data from the People, Animals, and their Zoonoses (PAZ) project to demonstrate these techniques: data collected from 415 homesteads in western Kenya, contained over 1500 variables that describe the health, environment, and social factors of the humans, livestock, and the homesteads in which they reside. The wide, sparse dataset was simplified to 42 predictors of P. falciparum malaria infection and wealth rankings were produced for all homesteads. The 42 predictors make biological sense and are supported by previous studies. This systematic data-mining approach we used would make many large datasets more manageable and informative for decision-making processes and health policy prioritization

Measurement of dσ/dyd\sigma/dy of Drell-Yan e+e−e^+e^- pairs in the ZZ Mass Region from ppˉp\bar{p} Collisions at s=1.96\sqrt{s}=1.96 TeV

Submitted to Phys. Letter BWe report on a CDF measurement of the total cross section and rapidity distribution, $d\sigma/dy$, for $q\bar{q}\to \gamma^{*}/Z\to e^{+}e^{-}$ events in the $Z$ boson mass region ($66M_{ee}We report on a CDF measurement of the total cross section and rapidity distribution, dσ/dy, for γ*/Z→e+e− events in the Z boson mass region (66<Mee<116 GeV/c2) produced in p pbar collisions at \sqrt{s}=1.96 TeV with 2.1 fb−1 of integrated luminosity. The measured cross section of 257±16 pb and dσ/dy distribution are compared with Next-to-Leading-Order (NLO) and Next-to-Next-to-Leading-Order (NNLO) QCD theory predictions with CTEQ and MRST/MSTW parton distribution functions (PDFs). There is good agreement between the experimental total cross section and dσ/dy measurements with theoretical calculations with the most recent NNLO PDFs.Peer reviewe