Moderate Heat-Assisted Gene Electrotransfer as a Potential Delivery Approach for Protein Replacement Therapy through the Skin

Gene-based approaches for protein replacement therapies have the potential to reduce the number of administrations. Our previous work demonstrated that expression could be enhanced and/or the applied voltage reduced by preheating the tissue prior to pulse administration. In the current study, we utilized our 16-pin multi-electrode array (MEA) and incorporated nine optical fibers, connected to an infrared laser, between each set of four electrodes to heat the tissue to 43 °C. For proof of principle, a guinea pig model was used to test delivery of reporter genes. We observed that when the skin was preheated, it was possible to achieve the same expression levels as gene electrotransfer without preheating, but with a 23% reduction of applied voltage or a 50% reduction of pulse number. With respect to expression distribution, preheating allowed for delivery to the deep dermis and muscle. This suggested that this cutaneous delivery approach has the potential to achieve expression in the systemic circulation, thus this protocol was repeated using a plasmid encoding Human Factor IX. Elevated Factor IX serum protein levels were detected by ELISA up to 100 days post gene delivery. Further work will involve optimizing protein levels and scalability in an effort to reduce application frequency.

Etranacogene Dezaparvovec (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B: Two Year Data from a Phase 2b Trial

Background: Gene therapy for hemophilia offers the possibility to ameliorate disease severity to a mild or functionally curative state through a single administration. Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter. Aims: We have previously shown a single dose of etranacogene dezaparvovec to provide sustained FIX activity into the mild-to normal range for up to 52 weeks post-dose in participants with severe or moderate-severe hemophilia B. This time, 2 years of follow-up data will be presented for the first time. Methods: A Phase 2b, open-label, single-dose, single-arm, multi-center trial (NCT03489291) in adult hemophilia B subjects. Interestingly, participants were not excluded based on neutralizing antibodies to AAV5. All subjects received a single intravenous dose of etranacogene dezaparvovec (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly to a mean of 31% at Week 6. At Week 52, mean FIX activity increased further to 41% with FIX activity levels of 50%, 31% and 41% in participants 1-3 respectively. There was no relationship between the presence of anti-AAV5 NAbs and response to etranacogene dezaparvovec. As of 52 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to etranacogene dezaparvovec and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 2 years of follow-up will be presented with the main focus on FIX activity, FIX replacement therapy use and reported bleeds. Conclusions: Patients with AAV5 NAbs were included in the Phase 2b etranacogene dezaparvovec trial and have shown sustained FIX activity into the mild-to normal range. All participants were able to discontinue routine prophylaxis, and there have been no bleeds post-treatment with etranacogene dezaparvovec. Disclosures Giermasz: BioMarin: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, Research Funding, Speakers Bureau; uniQure: Consultancy, Research Funding; Sangamo Therapeutics: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding, Speakers Bureau. Castaman:Alexion: Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Baxalta/Shire: Honoraria; Bayer: Honoraria; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kedrion: Speakers Bureau; Werfen: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau. Key:Novo Nordisk: Other: Chair of Grants Committee; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy. Miesbach:Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Recht:Spark: Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; uniQure: Consultancy, Other: personal fees, Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Pfizer: Consultancy, Other: personal fees; Genentech: Consultancy, Other: personal fees, Research Funding; CSL Behring: Consultancy, Other: personal fees. Gomez:Global Blood Therapeutics: Speakers Bureau. Gut:uniQure: Current Employment. Pipe:Siemens: Research Funding; Medical and Scientific Advisory Council to the National Hemophilia Foundation; Medical Advisory Board to World Federation of Hemophilia: Membership on an entity's Board of Directors or advisory committees; Apcintex, Bayer, BioMarin, Catalyst Biosciences, CSL Behring, HEMA Biologics, Freeline, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, Inc., Sangamo Therapeutics, Sanofi, Takeda, Spark Therapeutics, uniQure: Consultancy. OffLabel Disclosure: Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter.

One Year Data from a Phase 2b Trial of AMT-061 (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B

Background: Gene therapy for hemophilia offers the possibility of ameliorating the disease severity to a milder or functionally curative state through a single treatment. AMT-061 is an investigational gene therapy for hemophilia B comprising an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver-specific promoter. Aims: Confirm that a single dose of AMT-061 will provide a minimum-therapeutic response of FIX activity 6-weeks post-dose in participants with severe or moderate-severe hemophilia B. Here, 1 year of follow-up will be presented for the first time. Methods: Phase 2b, open-label, multi-center trial (NCT03489291) in adult males with FIX ≤2% and without active hepatitis or uncontrolled HIV. Participants were not excluded based on neutralizing antibodies to AAV5. Participants received a single intravenous dose of AMT-061 (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient-reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly (Figure) to a mean of 31% at Week 6. At Week 36, mean FIX activity increased further to 45% with FIX activity levels of 54%, 30% and 51% in participants 1-3 respectively. As of 36 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to AMT-061 and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 52 weeks of follow-up will be presented. Conclusions: Sustained elevation of FIX activity into the mild-to-normative range were observed in all participants 36 weeks after treatment with AMT-061. AMT-061 was safe and well-tolerated with no requirement for immunosuppression. These data support the ongoing Phase 3 study. Figure Disclosures Pipe: Novo Nordisk: Consultancy; Apcintex: Consultancy; Roche/Genentech: Consultancy; BioMarin: Consultancy; Shire: Consultancy; Sanofi: Consultancy; uniQure: Consultancy; Pfizer: Consultancy; HEMA Biologics: Consultancy; Catalyst Bioscience: Consultancy; Freeline: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Spark Therapeutics: Consultancy. Giermasz:Genentech/Roche: Consultancy, Other: Research, Speakers Bureau; Sangamo: Other: Research; BioMarin: Consultancy, Other: Research; uniQure: Consultancy, Other: Research; Bioverativ/Sanofi: Consultancy, Speakers Bureau. Castaman:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda (SHIRE): Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Uniqure: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Werfen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Key:Uniqure BV: Research Funding. Leebeek:CSL Behring: Research Funding; Baxalta/Shire: Research Funding; uniQure BV: Consultancy, Research Funding. Miesbach:Bayer, Chugai, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire, UniQure: Speakers Bureau; Bayer, BioMarin, CSL Behring, Chugai, Freeline, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda/Shire, UniQure: Consultancy; Bayer, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire: Research Funding. Recht:Bioverativ, CSL Behring, Genentech, Kedrion, NovoNordisk, Pfizer, Shire, Uniqure: Consultancy; American Thorombosis & Hemostasis Network: Other: Immediate Past Chair; Bioverativ, Genentech, NovoNordisk Shire: Research Funding. Gomez:Alnylam: Consultancy; Novo Nordisk, Novartis, Pfizer, Sanofi, Takeda, UniQure: Research Funding. Long:Uniqure BV: Employment. Gut:Uniqure BV: Employment. von Drygalski:University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.