Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer

Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

Meta-analysis of HIV-1 vaccine elicited mucosal antibodies in humans

We studied mucosal immune responses in six HIV-1 vaccine trials investigating different envelope (Env)-containing immunogens. Regimens were classified into four categories: DNA/vector, DNA/vector plus protein, protein alone, and vector alone. We measured HIV-1-specific IgG and IgA in secretions from cervical (n = 111) and rectal swabs (n = 154), saliva (n = 141), and seminal plasma (n = 124) and compared to corresponding blood levels. Protein-containing regimens had up to 100% response rates and the highest Env-specific IgG response rates. DNA/vector groups elicited mucosal Env-specific IgG response rates of up to 67% that varied across specimen types. Little to no mucosal IgA responses were observed. Overall, gp41- and gp140-specific antibodies dominated gp120 mucosal responses. In one trial, prior vaccination with a protein-containing immunogen maintained durability of cervical and rectal IgG for up to 17 years. Mucosal IgG responses were boosted after revaccination. These findings highlight a role for protein immunization in eliciting HIV-1-specific mucosal antibodies and the ability of HIV-1 vaccines to elicit durable HIV-1-specific mucosal IgG.

Widespread and sustained target engagement in Huntington’s disease minipigs upon intrastriatal microRNA-based gene therapy

Huntingtin (HTT)–lowering therapies hold promise to slow down neurodegeneration in Huntington’s disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging–guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 1013 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic.

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta

Objective: Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P <0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice ( P ≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. Conclusions: Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta.

Vector Shedding and Blood Biodistribution in Patients with Severe Hemophilia a Following Administration of Valoctocogene Roxaparvovec

Introduction: Long-term durable expression of hFVIII-SQ has been observed following BMN 270 (AAV5-hFVIII-SQ, valoctocogene roxaparvovec) single-dose administration in patients with severe hemophilia A. Although adeno-associated virus (AAV) vectors are replication incompetent and thus pose minimal risk for transmission or release into environment, a comprehensive assessment of vector shedding in secreta and excreta is required as part of the clinical development program. In addition, evaluation of vector biodistribution in blood is useful to characterize vector DNA processing and further understand the kinetics of vector DNA clearance. Vector shedding and biodistribution were evaluated from subjects from an ongoing Phase 1/2 study (Study 270-201, NCT02576795) and an ongoing Phase 3 study (Study 270-301, NCT03370913) following BMN 270 administration in patients with severe hemophilia A. Methods: In the Phase 1/2 study, 15 adult male subjects with severe hemophilia A received a single intravenous infusion of 6E12 vg/kg (n=1), 2E13 vg/kg (n=1), 4E13 vg/kg (n=6), or 6E13 vg/kg (n=7) BMN 270. In the Phase 3 study, 134 adult male subjects with severe hemophilia A received a single intravenous infusion of 6E13 vg/kg BMN 270. In both studies, measurement of vector DNA in blood, saliva, feces, semen, and urine was performed using a validated qPCR assay. Blood, saliva, urine, stool, and semen were collected until at least 3 consecutive negative results via qPCR were obtained. To further characterize vector DNA potentially capable of cell transduction, a novel immunocapture qPCR (iqPCR) assay was developed to measure the amount of intact AAV5 vector capsids in plasma and semen. Further assessments of the biodistribution of vector DNA in blood, including the evaluation of the contiguity and structural characteristics of BMN 270 vector genomes, were performed in blood, plasma, peripheral blood mononuclear cells (PBMC), and red blood cells using a drop-phase droplet-digital (dd)PCR assay. Results: Following BMN 270 administration at all dose levels, vector DNA was detected in all subjects in all biodistribution and shedding matrices evaluated (i.e., blood, saliva, urine, stool, and semen). Median peak vector DNA levels were greatest in blood followed by saliva, semen, stool, and urine. Peak vector DNA concentrations following BMN 270 administration were observed early. Following peak vector DNA concentrations, BMN 270 vector genomes were steadily cleared from the urine, semen, saliva, stool, and blood. In comparison to total vector DNA measured by qPCR, encapsidated vector DNA in plasma and semen was cleared more rapidly, as measured using iqPCR. Evaluation of total vector DNA in whole blood and blood fractions, indicate 3 phases of vector DNA clearance, which are associated with the expected lifespan of various transduced cell types. From approximately 24 weeks after BMN 270 administration and beyond, a slower rate of decline of vector DNA in whole blood is observed with the majority of transgene DNA present beyond 24 weeks in blood likely within the PBMC fraction. Further characterization of vector DNA in blood demonstrated that BMN 270 DNA transitioned from an initial truncated form into full-length transgenes over time. In addition, the fraction of DNA detected in whole blood that contains an inverted terminal repeat (ITR) fusion, indicating that the residual vector DNA may have formed circular episomes in the transduced cells, increased over time. By 52 weeks post-BMN 270 administration, the majority of vector DNA in whole blood was full-length and contained an ITR fusion. Conclusions: Vector shedding and distribution has been extensively evaluated in patients with severe hemophilia A treated with BMN 270. Both vector DNA and vector capsids were detected and steadily cleared in blood and shedding matrices. Based upon the replication incompetent nature of BMN 270 and the maximum potential exposure to the vector in secreta and excreta following BMN 270 administration, the risk of transmission to untreated individuals is considered extremely low. The biodistribution and characterization of vector DNA in blood cells demonstrates the formation of full-length transgenes with ITR fusions. Disclosures Clark: BioMarin Pharmaceutical In.: Current Employment. Hammon:BioMarin Pharmaceutical Inc.: Current Employment. Sandza:BioMarin Pharmaceutical Inc.: Current Employment. Torres:BioMarin Pharmaceutical Inc.: Current Employment. Koziol:BioMarin Pharmaceutical Inc.: Current Employment. Holcomb:BioMarin Pharmaceutical Inc.: Current Employment. Kim:BioMarin Pharmaceutical Inc.: Current Employment. Jayaram:BioMarin Pharmaceutical Inc.: Current Employment. Russell:BioMarin Pharmaceutical Inc.: Current Employment, Current equity holder in publicly-traded company; Amgen nc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Vettermann:BioMarin Pharmaceutical Inc.: Current Employment. Henshaw:BioMarin Pharmaceutical Inc.: Current Employment.

Broad Protection of Pigs against Heterologous PRRSV Strains by a GP5-Mosaic DNA Vaccine Prime/GP5-Mosaic rVaccinia (VACV) Vaccine Boost

Background: Porcine reproductive and respiratory syndrome (PRRS) viruses are a major cause of disease and economic loss in pigs worldwide. High genetic diversity among PRRSV strains is problematic for successful disease control by vaccination. Mosaic DNA and vaccinia (VACV) vaccines were developed in order to improve protection against heterologous PRRSV strains. Methods: Piglets were primed and boosted with GP5-Mosaic DNA vaccine and recombinant GP5-Mosaic VACV (rGP5-Mosaic VACV), respectively. Pigs vaccinated with rGP5-WT (VR2332) DNA and rGP5-WT VACV, or empty vector DNA and empty VACV respectively, served as controls. Virus challenge was given to separate groups of vaccinated pigs with VR2332 or MN184C. Necropsies were performed 14 days after challenge. Results: Vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains. In contrast, vaccination of animals with the GP5-WT vaccines induced responses only to VR2332. Furthermore, vaccination with the GP5-Mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (PAMs), and bronchoalveolar lavage (BAL) fluids, and less severe lung lesions after challenge with either MN184C or VR2332, which have only 85% identity. In contrast, significant protection by the GP5-WT based vaccines was only achieved against the VR2332 strain. Conclusions: GP5-Mosaic vaccines, using a DNA-prime/VACV boost regimen, conferred protection in pigs against heterologous viruses.

Clearance of Vector DNA Following Systemic Administration of AAV5-hFIX or AAV5-hFIX Padua in Patients with Severe or Moderate-Severe Hemophilia B

Introduction Current adeno-associated viral (AAV) vector-based gene therapy strategies for hemophilia rely on systemic administration of the vector. Durable expression of the transgene over the span of years has been reported from several trials, yet information on the clearance of vector material from bodily fluids is still limited and monitoring of "shedding" is required during trials despite lack of evidence of environmental or transmission risk. Here, we examined the magnitude and duration of the presence of vector DNA in participants from a Phase I/II study of an AAV5-hFIX wildtype construct (AMT-060; NCT02396342) and from a Phase IIb study utilizing the enhanced version, AAV5-hFIX Padua (AMT-061; NCT03489291). Methods Adult male participants with severe or moderately severe hemophilia B received a single intravenous infusion of either AMT-060 at 5x1012 genome copies(gc)/kg (low dose) or 2×1013 gc/kg (high dose), or AMT-061 at 2x1013gc/kg in one of two ongoing trials. Assessments in both trials included efficacy and safety outcomes as well as vector shedding in whole blood and semen. Vector shedding was also measured in nasal secretions, feces, urine, and saliva in participants receiving AMT-060. Vector shedding was analyzed using a validated quantitative real time polymerase chain reaction (qPCR) based assay measuring presence of vector DNA in the bodily fluids. Vector clearance was reached when vector DNA was either zero or below the limit of detection (LOD) for three consecutive measurements. The range in time to the first and the third consecutive negative measurement for each dose group are provided. Results Treatment with AMT-060 resulted in sustained improvement in FIX activity for up to 3.5 years [Mean FIX activity was 5.1% (low dose group at 3.5 years) and 7.5% (high dose group at 3 years)] and treatment with AMT-061 resulted in mean FIX activity of 45% over 36 weeks. AMT-060 and AMT-061 reduced the mean number of annualized bleeds by between 82% and 100% respectively. AMT-060 reduced the requirement for exogenous FIX administration by 86% and was abrogated with AMT-061. Both AAV5-hFIX and AAV5-hFIX Padua were safe and well tolerated and no unexpected TRAEs have been observed with longer-term follow up. Table 1 describes the time in weeks to the first and last of three consecutive measures of vector DNA of either zero or <LOD for all bodily fluids for AMT-060 and AMT-061. AMT-060 at the higher dose was cleared from semen, feces, urine, nasal secretions and saliva in all participants by week 64 (range 7-64 weeks). In blood, the lower dose of AMT-060 was cleared in all participants by 3 years (range 1.0-3.0 years). The higher AMT-060 dose was cleared in 4 of 5 participants by 2.5 years (range 1.8-2.5 years), and while below the LOD in the 5th participant by year 3, had not achieved the definition of vector clearance. With AMT-061, vector DNA were <LOD or below the lower limit of quantification in blood in all 3 participants by weeks 36-40 and in semen by week 26 in 2 of 3 participants but had not achieved the definition of vector clearance. Conclusions Post-AMT-060 treatment, vector DNA was undetectable in all participants in the high dose group by 10 months and considered cleared by 16 months in all bodily fluids except blood. AMT-060 was cleared from the blood in 100% of participants in the low dose group at 3 years and 80% of participants in the high dose group by 2.5 years. As expected, AMT-061 vector DNA was detectable at 36 weeks in blood and in the semen of 1 of 3 participants, although clearance had not yet been established in the remaining participants. The presence of vector DNA in bodily fluids assessed was not associated with any adverse safety or efficacy findings. Disclosures Sawyer: Uniqure BV: Employment. Gielen:uniQure Biopharma: Employment. Twisk:uniQure Biopharma: Employment. Long:Uniqure BV: Employment. Gut:Uniqure BV: Employment.