• ClinicalTrials.gov Identifier: NCT01687608¹
• A Phase 1/2 Open-Label, Single Ascending Dose Trial¹

• Active, not recruiting*, Last Update Posted: October 26, 2021¹
• Discontinued according to the presentation from the ISTH 2015
• Last Update Posted: September 30, 2022

• Males age 18-75 years, inclusive
• Established hemophilia B with ≥3 hemorrhages per year 
• Plasma FIX activity ≤2% (<1% for first cohort; then per protocol)¹

Detectable AAV8 neutralizing antibodies

AAV8/ HEK293 cells^2, 3

Statements regarding the utilized Codon-optimized FIX-Padua transgene were collected from here² and summarized as follows:

• The optimization algorithm led to elevated CpG density resulting in an increase of 19 to 99 CpG motifs in the FIX cDNA
• These CpG clusters may activate the innate immune system viaToll-like receptor 9 (TLR9)

The expression cassette in the self-complementary AAV vector includes the following regulatory elements²:

• Liver-specific murine transthyretin (TTR) enhancer/promoter
• The intron fragment from minute virus of mice (MVM) 
• And the bovine growth hormone (BGH) polyadenylation (pA) signal

Systemic⁽⁴²⁸⁾

Co. 1: 2e11 (2)
Co. 2: 1e12 (4)
Co. 3: 3e12 (2)

7 weeks to 2 years⁵ and up to 4-7.2 years^2, 3

OS ^2, 3

^a All 4 pts. in Co. 2 experienced significant levels of FIX activity; however, there was considerable inter-participant variability (Figure 2 ³⁴)
^b P5 achieved FIX activity of 20% at W7 and was the only participant with sustained activity from week 2 to 52 post-infusion. FIX activity
   was sustained during4 years’ follow-up. Although P3 and P4 lost 80% of peak expression acutely (over 2 weeks), neither exhibited increased
   liver transaminase levels, vector- or transgene-specific T-cell responses
^c Following their FIX peak expressions, P6 and P7 experienced a steep fall in FIX activity by W7 and W6.  Both pts. achieved a peak of ALT levels
  at W7 and an increase in AAV capsid–directed T-cell activation was observed at W6 before the FIX drop. Also, an increase and slight elevation
  in liver transaminase levels were observed at W7 in P6 and at W6 in P7

Further follow-up data regarding the transgene expression levels were collected from the abstract and the corresponding poster presented at the
64^th American Society of Hematology (ASH) Meeting in year 2022 ⁸³ and summarized as follows:

• 7/8 pts. did not achieve clinically meaningful levels of durable transgene expression despite the use of glucocorticoids. Most pts. resumed FIX replacement by 52 weeks
• P5 previously demonstrated high levels of durable FIX expression with no bleeds or need for replacement therapy for the first 4 years after BAX 335 administration
• This pt. had a FIX activity of 0.078 IU/ml (around 7.8%) after 7.2 years follow-up and was found to have a missense variant (c.344A>C, p.A115E) in the IL6R gene

According Figure 2 and Figure 3 published here² the peak factor response is achieved after 5 to 8 weeks post infusion

• 1/8 pts. achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for up to 7.2 years³
• In others, FIX activity was not sustained beyond 5 to 11 weeks²

Not reported

• Date concerning the IRRs were collected from here² and summarized as follows:

• Co. 1: 1/2 pts. feeling flushed (grade 1 hypersensitivity reaction) AE resolved within 30 minutes
• Co. 3: 1/2 pts. experienced asymptomatic elevation of serum IL-6 levels between 30 minutes and 8 hours that returned to baseline levels 24  hours post infusion

^a   Tiredness (mild)
^b   Exercise-induced rhabdomyolysis (severe)
^c   Feeling    flushed (mild), Influenza-like symptoms (mild), Ankle swelling (mild), elevated liver levels (moderate)
^d   Bacterial infection of right tonsil (moderate), Tonsillar hemorrhage (moderate)
^e   High blood pressure (mild) Left arm abscess (moderate)
^f   Tonsillar carcinoma (severe)
^g   Elevated liver enzyme levels (mild)

Further follow-up AEs data were collected from the poster presented at the 64th American Society of Hematology (ASH) Meeting in year 2022 83 and summarized as follows:

• Of 176 AEs, 126 (72%) occurred in years 1 and 2
• All SAEs resolved, and none were considered to be related to BAX 335
• 4 SAEs were reported at the planned 12-month analysis: rhabdomyolysis, bacterial tonsillitis, tonsillar hemorrhage, and squamous cell carcinoma of the tonsil1
• The stage IVA tonsillar squamous cell carcinoma occurred in 1 participant 8 months after infusion
• 2 additional SAEs (post-procedural hemorrhage and arthritis) and 2 additional possibly related AEs (localized edema and laryngeal dysplasia) occurred at data cut-off , May 6, 2022
• No FIX inhibitors were observed during the study

Not reported

ALT elevation peak levels were extracted from Figure 2 and Figure 3 from this publication²

• Co. 2: 25 – 45 IU/L, earliest peak noted at week 7 except for P5 with an ALT level peak at week 2 
• Co 3:  50 – 100 IU/L at week 7

AST elevation peak levels were extracted from Figure 2 and Figure 3 from this publication²

• Co. 2: 20 – 45 IU/L, earliest peak noted at week 4 except for P5 with an AST level peak at week 2
• Co 3:  30 – 60 IU/L at week 7

Capsid-directed T-cell activation peaks are extracted from Figure 2 and Figure 3 in this publication²

• Co. 2: only low elevations in the range of 80 - 100 SFU/1e6 PBMC with peaks at weeks 1, 10 and 6 were observed in 3/4 pts.    
• Co. 3: 700 – 800 SFU/1e6 PBMC with peaks at week 3 and 7 were observed in 2/2 pts. 

Data concerning immune responses and steroid treatments post-BAX 335 infusion were collected from here² and summarized as follows:  

• 2/8 pts., both in the highest dose cohort (Co. 3: 3e12 vg/kg), had strong IFN-g ELISpot signals for circulating AAV8 capsid–reactive T cells accompanied by elevated ALT/AST levels (Figure 3).
• Although corticosteroid therapy was associated with immediate normalization of the IFN-g ELISpot in P6, this signal remained elevated for weeks after the initiation of prednisone in P7.
• Systemic corticosteroid administration initiated in response to ALT elevations in P6 and P7, and as prophylaxis in P8, did not stabilize FIX activity levels in these pts. (Figure 2 and Figure 3).
• According Figure 2 and Figure 3, the duration of steroid treatment in these pts. was 12 – 20 weeks 

Data regarding a cancer in a pt. were taken from here² and summarized as follows: 

• Squamous cell carcinoma of the tonsil in P8 (Co. 2) was reported as SAEs
• BAX 335–specific quantitative PCR and integration site analysis by nonrestrictive linear amplification-mediated PCR^{2 [supplemental methods]} showed no evidence of AAV vector genomes in and/or integration into tonsillar tumor tissue
• All SAEs were therefore considered unrelated to BAX 335.
• The single SAE ongoing at the time of the interim analysis (tonsillar carcinoma) has since been resolved

Further follow-up data regarding the reported cancer case were collected from the poster presented at the 64^th American Society of Hematology (ASH) Meeting in year 2022³ and summarized as follows: 

• Risk factors in this participant included a 25 pack-years smoking history; the tissue biopsy was negative for HPV
• Genomic integration analysis on biopsy and healthy tissue was performed by nrLAM-PCR and deep sequencing
• No integration sites were detected in sample tissue and 2 uniquely mappable integration sites were identified in healthy tissue located next to genes not associated with malignancy. AAV vector was not detected in either tissue sample  

1. Open-Label Single Ascending Dose of Adeno-associated Virus Serotype 8 Factor IX Gene Therapy in Adults With Hemophilia B. Available at: Study Details | Open-Label Single Ascending Dose of Adeno-associated Virus Serotype 8 Factor IX Gene Therapy in Adults With Hemophilia B | ClinicalTrials.gov.
2. Konkle, B.A., et al., BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression. Blood, 2021. 137(6): p. 763-774. BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression | Blood | American Society of Hematology (ashpublications.org)
3. Escobar, M., et al., BAX 335 Hemophilia B Gene Therapy Phase 1/2 Clinical Trial: Long-Term Safety and Efficacy Follow-up. Blood, 2022. Volume 140 (Issue Supplement 1). BAX 335 Hemophilia B Gene Therapy Phase 1/2 Clinical Trial: Long-Term Safety and Efficacy Follow-up | Blood | American Society of Hematology (ashpublications.org)
4. Nathwani, A.C., Gene therapy for hemophilia. Hematology Am Soc Hematol Educ Program, 2019. 2019(1): p. 1-8. Gene therapy for hemophilia | Hematology, ASH Education Program | American Society of Hematology (ashpublications.org)
5. Monahan, P., et al., Update on a phase 1/2 open-label trial of BAX335, an adeno-associated virus 8 (AAV8) vector-based gene therapy program for hemophilia B. J Thromb Haemost, 2015. 13 (Suppl. 2): p. 87. Abstracts (nih.gov)

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