Rapid Characterization of AAV gene therapy vectors by Mass Photometry

Recombinant adeno-associated viruses (rAAV) are extensively used as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing, and large-scale vector production and purification methods have been developed. However, a better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of the rAAV preparations. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations, but also identify the partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or are time-consuming and require a large amount of material. MP can analyze sub-picomole quantities of sample, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor physical attributes of rAAV vectors.

Rapid determination of full and empty adeno-associated virus capsid ratio by capillary isoelectric focusing

: Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.

Recent advances in the analysis full/empty capsid ratio and genome integrity of adeno-associated virus (AAV) gene delivery vectors

: Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors,there are several quality control (QC)parameters that should be rigorously monitored to comply with clini-cal safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods,focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

How Computational Epitope Mapping Identifies the Interactions between Nanoparticles Derived from Papaya Mosaic Virus Capsid Proteins and Immune System

Background: Nanoparticles derived from plant viruses possess fascinating structures, versatile functions and safe properties, rendering them valuable for a variety of applications. Papaya mosaic Virus-Like Particles (VLPs) are nanoparticles that contain a repetitive number of virus capsid proteins (PMV-CP) and are considered to be promising platforms for vaccine design. Previous studies have reported the antigenicity of PMV nanoparticles in mammalian systems. Materials and Methods: As experiments that concern vaccine development require careful design and can be time consuming, computational experiments are of particular importance. Therefore, prior to expressing PMV-CP in E. coli and producing nanoparticles, we performed an in silico analysis of the virus particles using software programs based on a series of sophisticated algorithms and modeling networks as useful tools for vaccine design. A computational study of PMV-CP in the context of the immune system reaction allowed us to clarify particle structure and other unknown features prior to their introduction in vitro. Results: The results illustrated that the produced nanoparticles can trigger an immune response in the absence of fusion with any foreign antigen. Conclusion: Based on the in silico analyses, the empty capsid protein was determined to be recognised by different B and T cells, as well as cells which carry MHC epitopes.

AAV Purification by anion-exchange chromatography

We describe a new optimized, scalable and reproducible method based on anion exchange chromatography to obtain high titers of rAAV vectors without empty capsids contamination. The method takes advantage of Q-sepharose matriz to development a scalable procedure. After the virus harvest from supernatant and lysate cells, virus crude was subjected to anion exchange chromatography with Q-sepharose column. Three different protocols were tested, and the elution peaks were evaluated through qPCR rAAV titration and 260/280 nm ratio determination in order to identified empty capsid-containing fractions. A 150 mM NH4Ac wash step fallowing by 1 M NaCl elution step generate a a high titer eluted fraction of rAAV with 1.334 260/280 nm ratio. The described method makes rAAV vector purification an easily adapted for a large scale GMP production format to produce empty capsid free rAAV for clinics.

Recent progress in vaccines against foot-and-mouth disease

Foot-and-mouth disease (FMD) is the most highly contagious disease affecting livestock resulting in a significant adverse economic impact worldwide. Disease outbreaks in previously FMD-free countries are initially controlled by the culling of infected and in-contact animals, restriction of susceptible animal movement, and vaccination with an inactivated whole-virus antigen preparation. With increasing trade, commerce globalization, and people migration, it is likely that more inter-pool viral exchanges and spillovers will occur posing a greater threat to both, endemic and non-endemic regions, mostly due to the limited antigenic coverage of current vaccines. Currently available inactivated FMD vaccines have a number of disadvantages, including incomplete inactivation of the virus, they require multiple vaccination to maintain good levels of immunity and periodic inclusion of new viral strains into the vaccine formulation to cover new FMDV subtypes against which existing vaccines no longer protect and lack differentiation of infected from vaccinated animals (DIVA). The essential aim of DIVA strategy is realization of the so-called “vaccinate-to-live” policy, which is based on the principles that vaccinated animals exposed to FMDV will not transmit the virus. To address the shortcomings of inactivated vaccines, many efforts are currently devoted to developing novel FMD vaccines, including attenuated and marker inactivated vaccines, recombinant protein vaccines, synthetic peptide vaccines and empty capsid vaccines. Novel vaccine platforms offer promising alternatives for effective FMD control. It is likely that in the near future, multiple FMD vaccine approaches will compete for diverse markets, providing fit-for purpose solutions to evolving challenges in preventing and controlling FMD worldwide.

Adeno-Associated Mediated Gene Transfer for Hemophilia B:8 Year Follow up and Impact of Removing "Empty Viral Particles" on Safety and Efficacy of Gene Transfer

Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients. Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX <1%) were enrolled into the first and mid-dose cohorts, with six patients treated at the high dose. In a follow-on study arm, two severe HB subjects received a dose of 2x1012vg/kg of the new scAAV2/8-LP1-hFIXco preparation (full: empty capsid ratio 1:3) whilst the next 2 patients were treated at a dose of 5x1012vg/kg. In both arms, vector was administered without prophylactic immunosuppression but corticosteroids (starting at 60mg/day) were commenced if liver enzymes increased to ≥2 fold over baseline levels after gene transfer. Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids. Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. Disclosures Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

Novel Potent Capsid Assembly Modulators Regulate Multiple Steps of the Hepatitis B Virus Life Cycle

ABSTRACT The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies. Capsid assembly modulators (CAMs) induce the formation of empty capsid or aberrant capsid devoid of pregenomic RNA (pgRNA) and finally block relaxed circular DNA neosynthesis and virion progeny. In this study, the novel CAMs JNJ-827 and JNJ-890 were found to be potent inhibitors of HBV replication with respective half-maximal effective concentrations of 4.7 and 66 nM, respectively, in HepG2.117 cells. Antiviral profiling in differentiated HepaRG (dHepaRG) cells and primary human hepatocytes revealed that these compounds efficiently inhibited HBV replication, as well as de novo establishment of covalently closed circular DNA (cccDNA). In addition to these two known effects of CAMs, we observed for the first time that a CAM, here JNJ-827, when added postinfection for a short-term period, significantly reduced hepatitis B e antigen (HBeAg) secretion without affecting the levels of cccDNA amount, transcription, and hepatitis B surface antigen (HBsAg) secretion. This inhibitory activity resulted from a direct effect of JNJ-827 on HBeAg biogenesis. In a long-term treatment condition using persistently infected dHepaRG cells, JNJ-827 and JNJ-890 reduced HBsAg concomitantly with a decrease in viral total RNA and pgRNA levels. Altogether, these data demonstrate that some CAMs could interfere with multiple functions of HBc in the viral life cycle.