• ClinicalTrials.gov Identifier: NCT03369444¹
• A Phase I/II, Open Label, Ascending Single Dose Safety Study

• Terminated due to challenges during the COVID-19 pandemic and a change in requirements of data to be submitted for marketing authorization¹
• Last Update Posted: 2022-12-02¹

• Adults males, ≥ 18 years of age
• Severe FIX deficiency with plasma FIX activity of<1% of normal o rmoderately severe FIX deficiency with plasma FIX activity level ≥1% and ≤2%
• At least 150 exposure days to FIX concentrates

• Patients at high risk of thromboembolic events
• Further details concerning the exclusion of patients from treatment were published here², as follows:
  • Liver dysfunction in 1 patient
  • Presence or history of a factor IX inhibitor in 2 patients
  • Lack of a negative result on screening for AAVS3 neutralizing antibodies in 2 patients
  • Withdrawal from participation in the trial in 1 patient, and early termination of the trial in 2 patients
  • One patient had two reasons for exclusion, as follows:
    • A history of a FIX inhibitor and
    • Lack of a negative result on screening
      for AAVS3 neutralizing antibodies

Data regarding the serotype and manufacturing platform were collected form here^{2 [Supplementary Appendix]} and are summarized as follows:

• AAVS3 capsid that consists of a VP1u portion from AAV8 with the remainder (i.e. VP2u and VP3) from AAV3B
• Mammalian cell production system (HEK293T cell line)

FIX-Padua^2-3

New small liver‐ specific promoter referred to as FRE1^{2 [Supplementary Appendix], 3} or HLP2⁴
The following information regarding the FRE1 promoter was reported here² and is summarized as shown bellow:  

• The FRE1 promoter is a transcriptional control unit that consists of truncated elements of the
  • liver-specific human alpha-1 antitrypsin (hAAT) promoter and
  • human apolipoprotein E (ApoE) hepatic control region enhancer.
• The FIX Padua variant-encoding expression cassette^{2 [Supplementary Appendix]} contains the
  • Wild-type sequence of hFIX in exon 1 and the 5' portion of exon 2
  • These exons flank a truncated version of the intron natively present in that position
  • The 3' portion of exon 2 and the remaining exons are codon optimized and devoid of CpG dinucleotides
• The expression cassette contains also a bovine growth factor polyadenylation signal^{2 [Supplementary Appendix]}

Single IV infusion into a peripheral vein^{2 [Supplementary Appendix]}

Dose levels were adjusted according to a changed vector genome titer assay and reference standard^2, 5 as follows :

• Co. 1: 3.84e11 (2)
• Co. 2: 6.4e11 (2)
• Co. 3: 8.32xe11 (4)
• Co. 4: 1.28xe12 (2)

Details on follow-up times were published here² as follows:

• The primary end points were safety and efficacy, as assessed by FIX levels at week 26
• After 26 weeks, patients were enrolled in a long-term follow-up study

OS^2-3, FIX:C levels assessed by chromogenic assay were 2-fold lower³

• Steady state FIX:C levels in Co. 1 were 42.5 ± 6 % using an OS clotting assay³
• Following data regarding the mean FIX activity at W3 and W104 were taken from reference #6 and summarized as follows:
  • At W3, FIX activity levels in Co. 1- Co. 4 were in the range of 24% to 168 %
  • Pts. in Co. 1 (4.5e11vg/Kg), have stable, therapeutic, FIX activity levels through W104

Patient-specific FIX levels at early time points after gene therapy were reported here² and are summarized as follows:

• Co. 2 (6.4e11 vg/kg), prophylactic immunosuppression with prednisolone:
  • P4 had an initial steady increase in the FIX level to 47% by week 5
  • FIX levels in P4 decreased to less than 2% at month 11
• Co. 3 (8.32e11 vg/kg), prophylactic immunosuppression with glucocorticoids and tacrolimus:
  • FIX levels in P7 reached 228% at week 4 with steady levels with a mean of 52.3±2.9% for months 12 through 22
  • P8, P9, and P10 all had FIX levels of more than 150% by week 4 or 5
  • High FIX levels P8, P9, and P10 were sustained throughout the 26-week trial period
• Co. 4 (1.28e12 vg/kg), prophylactic immunosuppression with prednisolone:
  • FIX levels rose steadily in P3 and reached 167% by week 5
  • FIX levels in P6 increased into the normal range approximately 1 week after infusion and reached 310% at week 4
  • P6 required anticoagulation treatment with Apixaban for approx. 7 months

Further data on FIX levels per patient at week 26 and from month 12 trough the last follow-up after gene therapy have been published here² and are summarized as follows:

                   ^aPatient 4 resumed FIX prophylaxis in Month 13
                   ^bLess than 5 values available for calculation

According to Figure 2², the peak response in FIX activity was achieved around 5 to 10 weeks following gene therapy.

Follow-up data on annualized bleeding rates per patient have been published here² and are summarized as follows:

                ^aAnnualized data are based on the retrospective collection, three years prior to treatment
                ^bAnnualized data are from ≥15 days post-infusion to the last follow up available as of the data cut, no distinction is made between treated and untreated bleeds

Follow-up data on annualized FIX consumption per patient have been published here² and are summarized as follows:

                ^aAnnualized data are based on the retrospective collection, three years prior to treatment
                ^bAnnualized data are from ≥15 days post-infusion to the last follow up available as of the data cut, no distinction is made between treated and untreated bleeds
                ^cPatient 4 resumed FIX prophylaxis in Month 13

No evidence of infusion-related reactions⁵

• Observed FIX activities levels above 150% were individually assessed for risk of thrombosis, and one patient is being treated with DOACs⁵
• The most common drug related SAE was transient transaminitis (in 4 pts.) requiring supplemental immunosuppression⁶

AEs Related to FLT180a that have been reported during the 26-week B-AMAZE trial and the follow-up long-term study have been published here² and are summarized as follows:

               ^aIncluded in this category are increased levels of either alanine aminotransferase or aspartate aminotransferase

AEs Related to Glucocorticoids that have been reported during the 26-week B-AMAZE trial and the follow-up long-term study have been published here² and are summarized as follows:

• Co. 1 (3.84e11 vg/kg): No patient had increases in liver aminotransferase levels ^2-3

The detailed course of liver aminotransferase levels, specifically considering all participants in Co. 2 – Co. 4, was described here² as and is summarized follows:

• Co. 2 (6.4e11 vg/kg):
  • In P4, the ALT level increased from approx. 10 U per liter in weeks 1 to 4 to 57 U per liter in week 5.
  • A second increase in the ALT level in P4 occurred approx. 22 weeks after gene therapy
  • At week 4, P5 had a mild increase in the ALT level (39 U per liter)
• Co. 3 (8.32e11 vg/kg):
  • P7 had two episodes of increases in liver aminotransferase levels:
    • an initial breakthrough at week 5 and another
    • at week 16 after tapering of glucocorticoids
  • After the completion of tacrolimus, P8, P9 and P10 had increases in aminotransferase levels at or near month 6
• Co. 4 (1.28e12 vg/kg):
  • P3 was treated with intravenous methylprednisolone and tacrolimus for vector-related increases in ALT levels at week 7
  • In P6, at week 4, the ALT level had increased to a peak of 69 U/L

AST elevations were not reported explicitly

Not reported³

The detailed course of steroid treatment for all participants was described here² and is summarized as follows:

• Co. 1 (3.84e11 vg/kg): Prophylactic immunosuppression consisted of tapering courses of prednisolone starting at 60 mg daily from week 6 to week 12²
• Co. 2 (6.4e11 vg/kg) and Co. 4 (1.28e12 vg/kg):  
  • Prophylactic immunosuppression initiated with 60 - 90 mg of prednisolone daily between week 3 and week 4 didn’t prevent increases in ALT levels
  • Vector-related increases in ALT levels were responsive to intravenous methylprednisolone and tacrolimus

• Co. 3 (8.32e11 vg/kg): The immunosuppression regimen for P7 through P10 was amended to include prophylactic tacrolimus beginning alongside with glucocorticoids starting at week 3
  • Tacrolimus troughs in P7 were difficult to get into the desired range (10 to 15 ng/ml), possibly caused by a drug interaction with carbamazepine
  • P8, P9, and P10 all had FIX levels of more than 150% by week 4 or 5.
  • Prophylactic immunosuppression in P8, P9, and P10 with glucocorticoids and tacrolimus suppressed vector-related increases in liver aminotransferase levels,
    and high FIX levels were sustained throughout the 26-week trial period
  • P8, P9, and P10 received tacrolimus for 17 or 18 weeks with tacrolimus continued 6 to 8 weeks after cessation of glucocorticoids
  • After the completion of tacrolimus, all three pts. had increases in aminotransferase levels at or near month 6

According to Figure 2² and the corresponding results stated in that publication, certain ALT increases might be associated with a reduction in FIX transgene expression in the following participants:

• Co. 1: Not applicable as no participant had increases in liver aminotransferase levels^2-3
• Co. 2 (6.4e11 vg/kg), prophylactic immunosuppression with prednisolone:
  • In P4, whose FIX levels decreased to less than 2% by month 11, the first ALT peak level (57 U/L) at week 4 corresponded with a temporary reduction in its FIX levels from 47% to around 25%
• Co. 3 (8.32e11 vg/kg), prophylactic immunosuppression with glucocorticoids and tacrolimus:
  • The FIX levels in P7 decreased after the second episode of ALT elevation at week 16, dropped from about 220% to around 50%, which corresponded to a steady state from month 12 through month 22
• Co. 4 (1.28e12 vg/kg), prophylactic immunosuppression with prednisolone:
  • In P3, vector-related increases in ALT levels at week 7 were consistent with a decrease in FIX levels from 167% at week 5 to around 90% at week 9, as depicted in Figure 2

Not reported

1. A Factor IX Gene Therapy Study (FIX-GT) (FIX-GT).  Available at: Study Details | A Factor IX Gene Therapy Study (FIX-GT) | ClinicalTrials.gov
2. Chowdary P, Shapiro S, Makris M, Evans G, Boyce S, Talks K, Dolan G, Reiss U, Phillips M, Riddell A, Peralta MR, Quaye M, Patch DW, Tuddenham E, Dane A, Watissée M, Long A, Nathwani A. Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B. N Engl J Med. 2022 Jul 21;387(3):237-247. doi: 10.1056/NEJMoa2119913. PMID: 35857660. Phase 1–2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B | NEJM
3. Chowdary, P., et al., FLT180a: Next Generation AAV Vector for Haemophilia B - Long Term, Follow- up and In- depth Analysis of Transgenic FIX Using One- stage, Chromogenic and Global Assays. Res Pract Thromb Haemostasis, 2019. 3(S1): p. 307.ISTH Academy
4. Peyvandi, F. and I. Garagiola, Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia. Haemophilia, 2019. 25(5): p. 738-746.Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia - PubMed (nih.gov)
5. Chowdary, P., et al., Factor IX Expression within the Normal Range Prevents Spontaneous Bleeds Requiring Treatment Following FLT180a Gene Therapy in Patients with Severe Hemophilia B: Long-Term Follow-up Study of the B-Amaze Program. ASH Annual Meeting 2021, 2021. Oral and Poster Abstracts. Factor IX Expression within the Normal Range Prevents Spontaneous Bleeds Requiring Treatment Following FLT180a Gene Therapy in Patients with Severe Hemophilia B: Long-Term Follow-up Study of the B-Amaze Program - ScienceDirect
6. Chowdary, P., et al., A Novel Adeno Associated Virus (AAV) Gene Therapy (FLT180a) Achieves Normal FIX Activity Levels in Severe Hemophilia B (HB) Patients (B-AMAZE Study). ISTH 2020 Congress, 2020. Theme: Hemophilia and Rare Bleeding Disorders » Hemophilia Gene Therapy.

AAV, Adeno-associated virus; ABR, Annualized bleeding rate; AEs, adverse events; AIR, Annualized FVIII/FIX infusion rate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; 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; 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