Long-Term Follow-Up (LTFU) for Gene Therapy of Pyruvate Kinase Deficiency (PKD): A Phase I Clinical Trial to Evaluate the Safety of the Infusion of Autologous CD34+ Cells Transduced with a Lentiviral Vector Carrying the Codon Optimized Red Cell Pyruvate Kinase (coRPK) Gene in Adult and Pediatric Subjects with PKD

The primary objective of this Phase I clinical trial is to evaluate the **long-term safety** of the infusion of autologous CD34+ cells transduced with a lentiviral vector carrying the codon-optimized red cell pyruvate kinase (coRPK) gene in subjects with **pyruvate kinase deficiency** (PKD). This is clinically relevant as it aims to ensure the safety of a novel gene therapy approach, which could potentially offer a long-term solution for managing PKD, a condition characterized by chronic hemolytic anemia.

Additional primary objectives include:

• Determining the long-term persistence of the transgene in mononuclear cells in the blood and evaluating potential correlations between transgene persistence and transfusion independence, reduction in transfusion requirements, and clinically significant reduction of anemia.
• Assessing long-term clonality patterns beyond the RP-L301 parent study.
• Evaluating replication-competent lentivirus (RCL) in serum and peripheral blood cells, when relevant.

The clinical trial involves a total of **one** participant diagnosed with **pyruvate kinase deficiency**. The study population includes both male and female subjects, with an age range encompassing children and adolescents. Participants were selected based on their prior enrollment in the parent Study RP-L301-0119 and having received an autologous infusion of CD34+ enriched cells transduced ex vivo with a lentiviral vector carrying the coRPK gene. The trial does not involve a vulnerable population. Participants are required to adhere to the study visit schedule and other protocol requirements, having provided written informed consent. No specific lifestyle considerations such as diet or physical activity are highlighted for this study.

The clinical trial is designed to evaluate the long-term safety and efficacy of **autologous CD34+ hematopoietic stem cells** transduced ex vivo with a lentiviral vector encoding the codon-optimized version of the PKLR gene in patients with **pyruvate kinase deficiency**. This is a Phase I, long-term follow-up study, which is not categorized as low intervention. The trial employs a controlled design, with participants having previously been enrolled in the parent study RP-L301-0119. The primary objectives include assessing overall survival, peripheral blood genetic correction, and monitoring for any adverse events potentially related to the investigational product, RP-L301.

The trial is expected to run until July 31, 2038, with recruitment having commenced on January 1, 2023. Participants will be involved in the study for the duration of the trial, provided they meet the inclusion criteria, which require prior enrollment in the parent study and receipt of the investigational infusion. Participants must also be willing to adhere to the study visit schedule and protocol requirements. The study visits are structured to include an initial screening visit to confirm eligibility, followed by regular follow-up visits to monitor safety and efficacy outcomes. The end-of-study visit will conclude the participant's involvement, assessing long-term outcomes and any adverse events.

Participants may be withdrawn from the study if they experience significant adverse events, such as a sustained decrease in hemoglobin unrelated to infection or surgery, or if they develop any malignancy. Additionally, any adverse event considered at least possibly related to RP-L301 may lead to early termination from the study. The trial's primary endpoints focus on overall survival, genetic correction in peripheral blood, and the occurrence of any red blood cell transfusions. Secondary endpoints are not specified. The investigational product, Merilen, is administered via intravenous infusion, and the study is sponsored by Rocket Pharmaceuticals, Inc.

The clinical trial involves the administration of **Merilen**, an experimental gene therapy medicinal product (GTMP) developed by Rocket Pharmaceuticals, Inc. The active substance in Merilen consists of **autologous CD34+ hematopoietic stem cells** that have been transduced ex vivo with a lentiviral vector encoding the codon-optimized version of the PKLR gene. This product is designed to address Pyruvate Kinase Deficiency (PKD) by introducing a therapeutic human PKLR gene into the patient's own CD34+ cells. The pharmaceutical form of Merilen is an infusion, and it is administered via **intravenous infusion**. The dosing schedule and frequency of administration are determined based on the individual patient's condition and the study protocol. The product is not a pediatric formulation and is classified as an orphan drug under the designation EU/3/14/1330.

In addition to the experimental treatment, the study may involve the use of standard-of-care therapies as deemed necessary by the clinical investigators. These therapies are not specified in the trial data but are typically used to manage symptoms or complications associated with PKD. The trial does not include a placebo or comparator treatment, as the primary focus is on evaluating the long-term safety and efficacy of the gene therapy product. Participant compliance with the treatment regimen is monitored through regular follow-up visits and assessments, ensuring adherence to the study protocol and accurate evaluation of the treatment outcomes.

Efficacy in this clinical trial will be assessed through a series of primary endpoints designed to evaluate the therapeutic impact of the investigational product, **RP-L301**, in patients with Pyruvate Kinase Deficiency (PKD). The primary endpoints include overall survival, peripheral blood genetic correction as demonstrated by vector copy number (VCN), any red blood cell transfusion, sustained and significant decrease in hemoglobin unrelated to infection or surgery, adverse events related to worsening iron overload, any adverse event considered at least possibly related to RP-L301, and the development of any malignancy. These parameters will be measured and collected at specified intervals throughout the trial duration, allowing for a comprehensive analysis of the treatment's efficacy.