• 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 in Reference #2 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 Cohort 1, as reported in reference #3, are summarized below:

• Steady state FIX:C levels in Co. 1 were 42.5 ± 6 % using an OS clotting assay

The following data on mean FIX activity at Week 3 (W3) and Week 104 (W104) were taken from reference #6 and are 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 following gene therapy, as reported in reference #2, 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

Additional data on patient-specific FIX levels at Week 26 and from Month 12 through the last follow-up after gene therapy, as published in Reference #2, are summarized in the table below

             ^a Patient 4 resumed FIX prophylaxis in Month 13
             ^b Less than 5 values available for calculation

According to Figure 2 (Reference #2), 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 2 (Reference #2) 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 (References #2 and #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.  
2. A Factor IX Gene Therapy Study (FIX-GT). ClinicalTrials.gov identifier: NCT03369444. Link
3. Chowdary P, Shapiro S, Makris M, Evans G, Boyce S, Talks K, et al. 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. Link
4. 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 Haemost, 2019. 3(S1): p. 307. Abstract PB0309. Link
5. Peyvandi F, Garagiola I. Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia. Haemophilia. 2019 Sep;25(5):738-746. doi: 10.1111/hae.13816. Epub 2019 Jul 8. PMID: 31282050. Link
6. 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. Link
7. 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. Link
8.  

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