Development of novel lipoplex formulation methodologies to improve large-scale transient transfection for lentiviral vector manufacture.

Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.

Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography.

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion-exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV-1-LV recovery over quaternary-amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order-like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes ( t Y 1 / 2 ${t}_{{Y}_{1/2}}$ = 12.7 and 18.7 min). Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows.

Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing.

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Global Manufacturing of CAR T Cell Therapy.

Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient's blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial.

BACKGROUND: Parkinson's disease is typically treated with oral dopamine replacement therapies; however, long-term treatment leads to motor complications and, occasionally, impulse control disorders caused by intermittent stimulation of dopamine receptors and off-target effects, respectively. We aimed to assess the safety, tolerability, and efficacy of bilateral, intrastriatal delivery of ProSavin, a lentiviral vector-based gene therapy aimed at restoring local and continuous dopamine production in patients with advanced Parkinson's disease. METHODS: We undertook a phase 1/2 open-label trial with 12-month follow-up at two study sites (France and UK) to assess the safety and efficacy of ProSavin after bilateral injection into the putamen of patients with Parkinson's disease. All patients were then enrolled in a separate open-label follow-up study of long-term safety. Three doses were assessed in separate cohorts: low dose (1·9×10(7) transducing units [TU]); mid dose (4·0×10(7) TU); and high dose (1×10(8) TU). Inclusion criteria were age 48-65 years, disease duration 5 years or longer, motor fluctuations, and 50% or higher motor response to oral dopaminergic therapy. The primary endpoints of the phase 1/2 study were the number and severity of adverse events associated with ProSavin and motor responses as assessed with Unified Parkinson's Disease Rating Scale (UPDRS) part III (off medication) scores, at 6 months after vector administration. Both trials are registered at ClinicalTrials.gov, NCT00627588 and NCT01856439. FINDINGS: 15 patients received ProSavin and were followed up (three at low dose, six mid dose, six high dose). During the first 12 months of follow-up, 54 drug-related adverse events were reported (51 mild, three moderate). Most common were increased on-medication dyskinesias (20 events, 11 patients) and on-off phenomena (12 events, nine patients). No serious adverse events related to the study drug or surgical procedure were reported. A significant improvement in mean UPDRS part III motor scores off medication was recorded in all patients at 6 months (mean score 38 [SD 9] vs 26 [8], n=15, p=0·0001) and 12 months (38 vs 27 [8]; n=15, p=0·0001) compared with baseline. INTERPRETATION: ProSavin was safe and well tolerated in patients with advanced Parkinson's disease. Improvement in motor behaviour was observed in all patients. FUNDING: Oxford BioMedica.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

PURPOSE: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS: Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS: Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS: In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.

Safety and biodistribution of an equine infectious anemia virus-based gene therapy, RetinoStat(®), for age-related macular degeneration.

RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.

Dopamine gene therapy for Parkinson's disease in a nonhuman primate without associated dyskinesia.

In Parkinson's disease, degeneration of specific neurons in the midbrain can cause severe motor deficits, including tremors and the inability to initiate movement. The standard treatment is administration of pharmacological agents that transiently increase concentrations of brain dopamine and thereby discontinuously modulate neuronal activity in the striatum, the primary target of dopaminergic neurons. The resulting intermittent dopamine alleviates parkinsonian symptoms but is also thought to cause abnormal involuntary movements, called dyskinesias. To investigate gene therapy for Parkinson's disease, we simulated the disease in macaque monkeys by treating them with the complex I mitochondrial inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which induces selective degeneration of dopamine-producing neurons. In this model, we demonstrated that injection of a tricistronic lentiviral vector encoding the critical genes for dopamine synthesis (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, and guanosine 5'-triphosphate cyclohydrolase 1) into the striatum safely restored extracellular concentrations of dopamine and corrected the motor deficits for 12 months without associated dyskinesias. Gene therapy-mediated dopamine replacement may be able to correct Parkinsonism in patients without the complications of dyskinesias.

Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

The integration profile of EIAV-based vectors.

Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5' end of the transcription unit.

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

A decomposition model to track gene expression signatures: preview on observer-independent classification of ovarian cancer.

MOTIVATION: A number of algorithms and analytical models have been employed to reduce the multidimensional complexity of DNA array data and attempt to extract some meaningful interpretation of the results. These include clustering, principal components analysis, self-organizing maps, and support vector machine analysis. Each method assumes an implicit model for the data, many of which separate genes into distinct clusters defined by similar expression profiles in the samples tested. A point of concern is that many genes may be involved in a number of distinct behaviours, and should therefore be modelled to fit into as many separate clusters as detected in the multidimensional gene expression space. The analysis of gene expression data using a decomposition model that is independent of the observer involved would be highly beneficial to improve standard and reproducible classification of clinical and research samples. RESULTS: We present a variational independent component analysis (ICA) method for reducing high dimensional DNA array data to a smaller set of latent variables, each associated with a gene signature. We present the results of applying the method to data from an ovarian cancer study, revealing a number of tissue type-specific and tissue type-independent gene signatures present in varying amounts among the samples surveyed. The observer independent results of such molecular analysis of biological samples could help identify patients who would benefit from different treatment strategies. We further explore the application of the model to similar high-throughput studies.

The African swine fever virus protein j4R binds to the alpha chain of nascent polypeptide-associated complex.

The African swine fever virus (ASFV) j4R protein is expressed late during the virus replication cycle and is present in both the nucleus and the cytoplasm of infected cells. By using the yeast two-hybrid system, direct binding, and coprecipitation from cells, we showed that the j4R protein binds to the alpha chain of nascent polypeptide-associated complex (alpha NAC). Confocal microscopy indicated that a proportion of j4R and alpha NAC interact in areas close to the plasma membrane, as well as through the cytoplasm in cells. In vitro binding studies suggested that binding of j4R to alpha NAC did not interfere with the binding of alpha- and beta NAC subunits (the BTF3 transcription factor).

Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA.

Five independent clones of the Propionibacterium acnes P-37 lipase gene (gehA) were obtained in Escherichia coli, and the gene was localized to a 2.75 kb Xhol fragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe to gehA. The nucleotide sequence of gehA was determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabelled polypeptide was detected from E. coli minicell preparations harbouring gehA, which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide. gehA was overexpressed in E. coli under the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase from P. acnes culture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.