Terminated early due to challenges during the COVID-19 pandemic and a change in requirements of data to be submitted for marketing authorisation.

Severe haemophilia B (HB) is a bleeding disorder where a protein made by the body to help make blood clot is either partly or completely missing. This protein is called a clotting factor; with severe haemophilia B, levels of clotting factor IX (FIX) (nine) are very low and affected individuals can suffer life threatening bleeding episodes. HB mainly affects boys and men (normally one in every 30,000 males). Current treatment for HB involves intravenous infusions of factor IX as regular treatment (Prophylaxis) or 'on demand'. On demand treatment is highly effective at stopping bleeding but cannot fully reverse long-term damage that follows after a bleed. Regular treatment can prevent bleeding, however can be invasive for patients and also expensive. This research study aims to test the safety and effectiveness of a gene therapy which produces Factor IX protein in the body. The gene will be given using an inactivated virus called "the vector" ( FLT180a), in a single infusion. The vector has been developed from a virus known as an adeno- associated virus, that has been changed so that it is unable to cause a viral infection in humans. This "inactivated" virus is further altered to carry the Factor IX gene and to make its way within liver cells where Factor IX protein is normally made.

Up to three different doses cohorts of FLT180a will be tested, in up to 24 patients with severe haemophilia B. Patients will be recruited from haemophilia centres in the EU and US. Patients will be in the trial for approximately 40 weeks and will undergo procedures including physical examinations, bloods tests, ECGs and liver ultrasounds.

This was a Phase I/II, open-label, multicentre, ascending single-dose, safety study of FLT180a in patients with severe (FIX activity <1%) or moderately severe (FIX activity 1% to 2% with severe bleeding phenotype) HB. Up to 24 patients were planned to be enrolled; however, the study was terminated early (on 20 October 2020) after 10 patients had been enrolled because of changes to the clinical development plan and recruitment difficulties due to the COVID-19 pandemic.

Patients who provided consent to participate and had historical data on bleeding and FIX consumption documented from the previous 3 years' medical notes were screened for eligibility in this study. During the screening period, patients completed a diary to prospectively record ongoing bleeding events and FIX consumption. Patients were monitored through a comprehensive schedule of safety assessments at outpatient visits for 26 weeks.

On completion of the study, patients are to be followed for 15 years under a separate long term follow-up protocol.

Patients who provided consent to participate in this study underwent screening assessments up to 52 weeks before study Day 0 (FLT180a infusion). Due to the risk of bleeding in this patient population, a washout from the patient's FIX concentrate regimen was not mandated. The investigator was to demonstrate, from the patient's medical records, a documented FIX activity level of <1% for severe patients or <2% for moderately severe patients. If (at the investigator's discretion) a FIX concentrate washout was undertaken during the screening period, a minimum of 5 days' washout was required.

Treatment-eligible patients reported to the study site on the day before receiving the gene therapy infusion (Day 1). On Day 0, FLT180a was administered as a single dose, slow intravenous (IV) infusion into a peripheral vein. The patient remained in the study centre for ≥12 hours and until the investigator deemed the patient fit to be discharged. The first 2 patients treated at each dose level remained at the study centre for 24 hours after infusion before discharge.

Patients who were on prophylactic therapy with FIX concentrates remained on their usual dosing schedule and were closely monitored for FIX activity levels after screening and administration of FLT180a. If FIX activity levels ≥3% were reached, then prophylaxis was held pending a repeat analysis within a period of 72 hours. If the FIX activity levels were ≥3% at that time, then prophylaxis was stopped with continued/regular assessment of FIX activity levels and occurrence of spontaneous bleeding.

Patients were required to undergo study evaluations at intervals over the 26-week, post-treatment period. These evaluations took place either at the study infusion site or at their normal haemophilia treatment centre. To monitor for shedding of vector genome (vg) sequences, patients were required to provide plasma, saliva, urine, stool, and semen samples until the results of 3 successive samples were clear.

This was a first-in-human study; therefore, an ascending-dose design was implemented to enable dose evaluation in a step-wise manner. Three dose cohorts of vector (low, intermediate, and high) were tested in the dose escalation. Two patients were tested at each dose level with an additional patient added in the event of a dose limiting toxicity (DLT) (2 + 1 design). Dose escalation occurred provided there was no more than 1 DLT at any dose cohort and if the resulting FIX activity failed to reach the target level. A reduction of the dose level within a cohort occurred if the FIX activity exceeded defined levels to reduce the risk of exceeding the normal physiological range. A dose reduction occurred when the 2 + 1 design was applied at that new dose level within the cohort. At the discretion of the Sponsor after advice from the trial management group (TMG) and independent data monitoring committee (DMC), additional patients were to be added to any cohort to ensure adequate characterisation of either safety or the FIX response before dose escalation/reductions. The Sponsor, TMG, and DMC planned to select the terminal dose level based on the patient FIX activity levels with the aim of ensuring most patients reached a FIX activity level within normal limits and in the absence of DLTs; the terminal dose level was planned to be expanded to 14 patients, but never reached. This design minimised the number of patients who would need to be dosed at suboptimal levels while allowing evaluation of safety with the option to expand a group on observation of DLTs. An extended 6-week interval was observed between the first and second patient on study to monitor for any unanticipated, delayed adverse events (AEs). Subsequently, when dose escalation was ongoing, the study mandated a minimum 4-week interval between patients during which time efficacy and safety was reviewed before a decision to dose the next patient.

The main risk in this study was a dose-dependent, asymptomatic increase in the serum alanine aminotransferase (ALT) level associated with a decline in FIX levels, suggesting a loss of transduced hepatocytes. In this study, all patients were given a take-home pack of immunosuppressants (prednisolone only) to be taken under the direction of the investigator, which allowed rapid intervention if transaminase elevations were observed. In addition, during the anticipated critical time period all patients were to receive a course of immunosuppressants (prednisolone, methylprednisolone, and tacrolimus) beginning at the Week 3 visit or Week 4 visit in line with the relevant protocol version active at the time.

The main efficacy endpoint was based on an analysis of the proportion of patients achieving a clinical or normalised FIX response at 26 weeks. A clinical FIX response was defined as achieving a FIX activity of 5% to 150% of normal. Five percent had been selected as the threshold for a clinical FIX response because using gene therapy in patients with HB to increase FIX activity from <1% to 5% had previously been shown to lead to a highly clinically significant improvement in annualised bleeding rates (ABR) and exogenous factor consumption. A normalised FIX response was defined as achieving a FIX activity level in the normal range (50% to 150%). The normal range had been selected as the threshold level for a normalised FIX response because reaching this level was expected to modify the patient phenotype from severe at study start to normal at which point patients would not be expected to experience spontaneous bleeds.

The choice of a 26-week endpoint was based on previous experience with Adeno-Associated Virus (AAV) gene therapy for HB in which patients achieved steady-state FIX levels by 16 weeks after gene therapy. Based on this, it was anticipated that the patients' FIX activity would reach a stable level by 26 weeks, thus this was an appropriate point at which to measure activity.

Inclusion Criteria:

1. Adults males, ≥ 18 years of age.

2. Confirmed diagnosis of HB defined as one of the following:

   1. Documented severe FIX deficiency with plasma FIX activity of <1% of normal, or
   2. moderately severe FIX deficiency with plasma FIX activity level between ≥1% and ≤2% and a severe bleeding phenotype defined by one of the following: i. On prophylaxis for a history of bleeding, or ii. On demand therapy with a history of 4 or more bleeding episodes/year on average over the past 3 years, or iii. evidence of chronic haemophilic arthropathy (pain, joint destruction, and loss of range of motion).

3. Able to give full informed consent and able to comply with all requirements of the trial including 15-year long-term follow-up.

4. Willing to practice barrier contraception until at least 3 consecutive semen samples after vector administration are negative for vector sequences.

5. Lack of neutralising anti-AAV-S3 antibodies using an in vivo transduction inhibition assay within 4 weeks of vector administration.

6. At least 150 exposure days to FIX concentrates.

Exclusion Criteria:

1. Presence of neutralising anti-human FIX antibodies (inhibitor, determined by the Bethesda inhibitor assay) at the time of enrolment or a previous history of FIX inhibitor;
2. Patients at high risk of thromboembolic events (high risk patients would include those with a history of arterial or venous thromboembolism (e.g. deep vein thrombosis, pulmonary embolism, non-haemorrhagic stroke, arterial embolus) and those with acquired thrombophilia including conditions such as atrial fibrilation);
3. Use of investigational therapy for haemophilia within 30 days before enrolment;
4. Patients with active hepatitis B or C, and hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) Ribonucleic acid (RNA) viral load positivity, respectively, or currently on antiviral therapy for hepatitis B or C. Negative viral assays in 2 samples, collected at least 6 months apart, will be required to be considered negative. Both natural clearers and those who have cleared HCV on antiviral therapy are eligible.;
5. Serological evidence of human immunodeficiency virus (HIV-1);
6. Evidence of liver dysfunction (persistently elevated alanine aminotransaminase, aspartate aminotransferase, bilirubin >1.5 x upper limit of normal);
7. Platelet count <50 x 109/L;
8. Uncontrolled glaucoma, diabetes mellitus, or hypertension;
9. Malignancy requiring treatment;
10. Patients with uncontrolled cardiac failure, unstable angina or myocardial infarction in the past 6 months;
11. Poor performance status (World Health Organization score >1);
12. Prior treatment with any gene transfer medicinal product;
13. Known or suspected intolerance, hypersensitivity or contraindication to the investigational product and non-investigational medicinal products or their excipients;
14. Planned major elective surgery prior to the end of trial.
15. Current or relevant history of a physical or psychiatric illness or any medical condition that in the opinion of the investigator could affect the patients safety or interfere with the study assessments.
16. Cytomegalovirus (CMV) Immunoglobulin G (IgG) positive patients who are CMV PCR positive at screening.

From consent to Week 26 post dose/End of Study visit or early withdrawal of patient (up to 38 weeks).

Any untoward medical occurrence in a clinical investigation subject administered a pharmaceutical product that does not necessarily have a causal relationship with this treatment.

Protocol defined number of participants (n=24) was not met, the terminal dose as specified in the protocol was not identified prior to early study termination.

The primary efficacy endpoints (FIX response at the terminal dose) could therefore not be detailed established. However the FIX response data (efficacy endpoint) for the 4 patients treated at the last studied dose level 1.3×10e^12 vg/kg, have been presented instead.