• ClinicalTrials.gov Identifier: NCT03370913^1, 2 and NCT03392974³
• Phase III, Single-group, Open-label, GENEr8-1 (301; NCT03370913) and GENEr8-2 (302; NCT03392974) Trials

• GENEr8-1 (301; NCT03370913): Active, not recruiting, Last Update Posted: December 23, 2022¹
• GENEr8-2 (302; NCT03392974): After review of interim 301 results, enrollment into 302 was stopped⁴,  Last Update Posted: August 3, 2022³

• Male ≥18 years of age
• Base FVIII level ≤ 1 IU/dL⁵
• Treated/exposed to FVIII concentrates or cryoprecipitate for a minimum of 150 Eds

 Key exclusion criteria were reported here⁶ as follows:

• Anti-AAV5 capsid total binding antibodies
• HIV infection (added as a criterion after a protocol amendment)
• Substantial liver dysfunction, substantial liver fibrosis, or liver cirrhosis

AAV5/baculovirus, Spodoptera frugiperda (Sf9) insect-cell production system^{6 [supplementary appendix]}

Codon-optimized expression cassette for the SQ variant of B-domain–deleted hFVIII (AAV-HLP-FVIII-SQ)⁷

HLP is a 251-bp enhancer/promoter fragment containing 

• a 34-bp core enhancer from the human ApoE-HCR gene 
• a modified 217-bp gene promoter comprising the distal X and the proximal A1B regulatory domains from SERPINA1 (α-1-antitrypsin)⁸

The FVIII expression cassette also includes a synthetic polyA signal^{7 [supplementary material]}

    AAV5/baculovirus, Spodoptera frugiperda (Sf9) insect-cell production system^{6 [supplementary appendix]}

Three participants with HIV infection were enrolled in two Phase 3 trials as follows:

1. Trial: GENEr8-1 (301; NCT03370913)¹
   Enrollment: 2 participants with HIV (P1 and P2) that were included in previously reported
   safety results as part of the intent-to-treat population (ITT Pop. , N = 134)², but not in efficacy results
   Vector Dose: 6e13 vg/kg
2. Trial: GENEr8-2 (302; NCT03392974)³
   Enrollment: 1 participants with HIV (P3)
   Vector Dose: 4e13 vg/kg

According to the data shown in Figure 2⁴, the follow-up period for the study participants (P1 and P2) was as follows:

• P1 and P2: 174 weeks
• P3: 52 weeks, as reported on clinicaltrials.gov (NCT03392974)³

OS and CH⁴

• FVIII activity approximations for P1 and P2 over a span of 174 weeks were retrieved from Figure 1E-F⁴ and summarized in the table below
• Additionally, the last row of the table presents data on FVIII activity levels for P3, which are reported in the same publication⁴

Peaks were already reached at the earliest reporting time, which was at week 26 as depicted in Figure 1E-F⁴

 As indicated in the table bellow the treated ABR of P1 decreased from baseline, but P2 experienced bleeding episodes requiring treatment beyond week 105 as FVIII activity declined⁴

^a P2 experienced bleeding episodes requiring treatment beyond week 105 as FVIII activity declined
 

As indicated in the table bellow Valoctocogene roxaparvovec provided endogenous FVIII production and decreased annualized FVIII infusion rate for both pts.⁴

^a P2 experienced bleeding episodes requiring treatment beyond week 105 as FVIII activity declined
 

Not reported

     ^a Drug interactions between efavirenz-based HAART (highly active antiretroviral therapy) in P3 and valoctocogene roxaparvovec might lead to hepatocellular toxicity

Not reported

Data related to elevated ALT levels after Valoctocogene roxaparvovec infusion in participants with HIV were reported her⁴ and summarized as follows:

• P1 experienced no elevations in liver enzymes
• According Figure 1D, P2 experienced elevated ALT levels with two peaks between
  • Day 75 and day 100 (≈ 50 U/L) and
  • Day 100 and 125 (≈ 50 U/L) after infusion
• On day 34, P3 experienced grade 2 elevated ALT levels (Figure 1G)
• These ALT levels worsened to grade 3 SAE (peak of ≈ 800 IU/L, between day 50 and day 60)

Data related to elevated AST levels after Valoctocogene roxaparvovec infusion in participants with HIV were reported here⁴ and summarized as follows:

• P2 developed grade 1 elevated AST levels of 90 U/l between day 75 and day 100 after gene transfer
• The AST elevation in P2 resolved with prednisone
• P3 experienced grade 2 elevated AST levels (peak > 450 IU/L, between day 60 and day 70) (Figure 1G)

Not reported 

The responsiveness of prednisone to elevated liver enzymes was reported here⁴ and summarized bellow as follows: 

• In P3, the elevated ALT and AST levels  showed no response to prednisone treatment from day 34 to day 63
• On day 52, when the component of P3’s HAART^b regimen, efavirenz^a, was halted , the pause was associated with plateaued ALT levels
• However, upon reinstatement of efavirenz, ALT levels worsened again by day 63
• Within 4 days of replacing P3’s HAART regimen with a non-efavirenz-based HAART 
  • the liver enzyme tests dropped by approximately 25%
  • The improvement in liver enzyme levels continued until day 105 when the AEs of liver enzymes were considered resolved

^a Efavirenz is a nonnucleoside reverse transcriptase inhibitor (NNRTI) associated with hepatic injury
^b HAART, highly active antiretroviral therapy

The following statements concerning the decline in FVIII transgene expression, which might be associated with ALT elevation, are based on the data published here⁴ :

• In P1, the decline in FVIII transgene expression over the course of time from week 26 to week 104 was not associated with ALT elevation
• In P2, the decline in FVIII transgene expression was associated with mild elevation in ALT and grade 1 elevation in AST levels
• In P3, the participant with the highest liver enzymes elevations and efavirenz-based HAART, the correlation is not applicable due to the low FVIII expression from the beginning of the gene therapy

Not reported

1. Single-Arm Study To Evaluate The Efficacy and Safety of Valoctocogene Roxaparvovec in Hemophilia A Patients (BMN 270-301) (BMN 270-301). Available at: Study Details | Single-Arm Study To Evaluate The Efficacy and Safety of Valoctocogene Roxaparvovec in Hemophilia A Patients (BMN 270-301) | ClinicalTrials.gov
2. Ozelo, C.M., et al., Efficacy and Safety of Valoctocogene Roxaparvovec Adeno-associated Virus Gene Transfer for Severe Hemophilia A: Results from the Phase 3 GENEr8-1 Trial. ISTH 2021 Congress, 2021. Efficacy and Safety of Valoctocogene Roxaparvovec Adeno-associated Virus Gene Transfer for Severe Hemophilia A: Results from the Phase 3 GENEr8-1 Trial - ISTH Congress Abstracts
3. Single-Arm Study To Evaluate The Efficacy and Safety of Valoctocogene Roxaparvovec in Hemophilia A Patients at a Dose of 4E13 vg/kg (GENEr8-2). Available at: Single-Arm Study To Evaluate The Efficacy and Safety of Valoctocogene Roxaparvovec in Hemophilia A Patients at a Dose of 4E13 vg/kg - Full Text View - ClinicalTrials.gov
4. Ragni, M.V., et al., Valoctocogene roxaparvovec gene transfer in participants with HIV. Blood Adv, 2023. 7(8): p. 1525-1530. Valoctocogene roxaparvovec gene transfer in participants with HIV | Blood Advances | American Society of Hematology (ashpublications.org)
5. new C Ozelo M, Mahlangu J, Pasi K, Giermasz A, D Leavitt A, Laffan M, Symington E, V Quon D, Wang J-, Peerlinck K, Pipe S, Madan B, S Key N, F Pierce G, O’Mahony B, Kaczmarek R, Lawal A, Huang M, Wong WY, Kim B, GENEr8-1 Trial Group . Efficacy and Safety of Valoctocogene Roxaparvovec Adeno-associated Virus Gene Transfer for Severe Hemophilia A: Results from the Phase 3 GENEr8-1 Trial [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). Efficacy and Safety of Valoctocogene Roxaparvovec Adeno-associated Virus Gene Transfer for Severe Hemophilia A: Results from the Phase 3 GENEr8-1 Trial - ISTH Congress Abstracts
6. Ozelo, M.C., et al., Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A. N Engl J Med, 2022. 386(11): p. 1013-1025. Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A | NEJM
7. Rangarajan, S., et al., AAV5-Factor VIII Gene Transfer in Severe Haemophilia A. N Engl J Med, 2017. 377(26): p. 2519-2530. AAV5–Factor VIII Gene Transfer in Severe Hemophilia A | NEJM
8. McIntosh, J., et al., Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood, 2013. 121(17): p. 3335-44. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant | Blood | American Society of Hematology (ashpublications.org)

AAV, Adeno-associated virus; AEs, Annualized bleeding rate; AEs: adverse events; AIR, Annualized FVIII/FIX infusion rate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BL, baseline; CH, Chromogenic Assay; Co., cohort; DOACs, Direct oral anticoagulants; D, days; EDs, exposure days; FIX, factor IX; FIX-Padua, gain of function FIX variant; FVIII, factor VIII; gc, genome copies; HAART, highly active antiretroviral therapy; HEK cells, human embryonic kidney cells; IQR, interquartile range; IRR, Infusion-related reaction; NAbs, neutralizing antibodies; OS, One-stage clotting assay; Pop., population; pt., patient/participant; pts., patients/participants; P1, Participant 1; PI, phase I; PBGD, porphobilinogen deaminase; PBMC, peripheral blood mononuclear cells; SAEs, serious adverse events; SFU, spot-forming units; TAC, tacrolimus; ULN, upper limit of normal; VCN, vector copy number; vg, vector genomes; W, weeks; WT, wild type; Y, year