Adeno-Associated Virus Gene Therapy Translation: Lessons from Early Regulatory Meetings

Abstract

The Platform Vector-Gene Therapy (PaVe-GT) program is a National Institutes of Health (NIH) initiative that aims to develop adeno-associated virus (AAV) gene therapies for four monogenic rare diseases, two organic acidemias and two congenital myasthenic syndromes. PaVe-GT’s platform-based approach identifies and diminishes redundancies and applies efficiencies in preclinical, clinical, and regulatory activities. The program’s hypothesis is that implementing these efficiencies can accelerate clinical trial initiation. Based on its platform-centric experience and public-serving mission, the PaVe-GT program actively shares its scientific and regulatory learnings with the public to benefit the development of similar gene therapy products for rare diseases. PaVe-GT’s first investigational AAV gene therapy candidate is AAV serotype 9 human propionyl-CoA carboxylase alpha subunit (AAV9-hPCCA) for propionic acidemia caused by PCCA deficiency, which received initial feedback from the Food and Drug Administration (FDA) in an INitial Targeted Engagement for Regulatory Advice on CBER/Center for Drug Evaluation and Research (CDER) ProducTs (INTERACT) meeting. Upon further product development that took into consideration the FDA’s initial advice, the program obtained the Agency’s feedback in pre-investigational new drug (IND) (Type B) and Type C meetings. Here, we share our experience from these meetings, including strategy, preparation, pre- and post-meeting feedback from the FDA, and lessons learned during the AAV9-hPCCA regulatory process, which the program plans to apply across the PaVe-GT platform. Topics discussed in the regulatory meetings included animal model and efficacy studies, toxicology study plans, manufacturing of the investigational AAV product, and clinical trial design. The main lessons learned from the pre-IND and Type C meetings for AAV9-hPCCA are: (1) Pharmacology/Toxicology studies in a single rodent species are sufficient for filing an initial IND; (2) FDA feedback guides product quality improvements and early development of a quantitative potency assay; (3) use of biomarkers as potential surrogate endpoints in a future efficacy trial benefits from collection of data in the natural history study and the first-in-human Phase 1/2 study; and (4) evidence from the Phase 1/2 clinical trial could be leveraged to support a license application. Lightly redacted regulatory documents and comprehensive templates developed by the PaVe-GT team are available on the PaVe-GT website.

Keywords

rare diseases pre-IND meeting FDA type B meeting FDA type C meeting gene therapy PaVe-GT propionic acidemia PCCA AAV9

INTRODUCTION

Recombinant adeno-associated virus (AAV)-based gene therapy is a gene replacement strategy in which a functional copy of the mutated gene is introduced into living cells using an AAV vector. In the United States (U.S.), a milestone was achieved in 2017 with the U.S. Food and Drug Administration (FDA) approval of the first AAV gene therapy product, LUXTURNA^®, for confirmed biallelic RPE65 mutation-associated retinal dystrophy.¹ Six more AAV-based gene therapies were approved by the FDA for rare diseases by the end of 2024, and more than 200 AAV gene therapy trials for multiple indications are recorded in ClinicalTrials.gov.^2,3 Still, with greater than 10,000 rare diseases⁴ and only ∼5% of them having an FDA-approved treatment,⁵ there is a clear need for accelerated gene therapy development.

The Platform Vector-Gene Therapy (PaVe-GT) program is a National Institutes of Health (NIH) initiative to support the translation of AAV gene therapies for four rare diseases, two organic acidemias and two congenital myasthenic syndromes.⁶ The program’s goals are: (1) to determine whether a platform approach can define efficiencies and apply them across preclinical and clinical development to accelerate clinical trial startup and (2) to share with the public the lessons learned from the translation of the four AAV gene therapies. The PaVe-GT shared scientific and regulatory knowledge can also enable and de-risk the development of other gene therapy programs. The first investigational PaVe-GT candidate is AAV9-hPCCA (or NCATS BL-0746), an AAV9 vector carrying a human codon-optimized cDNA of PCCA for the treatment of propionyl-CoA carboxylase alpha subunit (PCCA)-related propionic acidemia (PA). PA is a rare monogenic disease with an estimated incidence of 0.13–1.2 infants per 100,000 births and no causal treatment available.^7–9 AAV9-hPCCA received early feedback from the FDA in an INitial Targeted Engagement for Regulatory Advice on CBER/CDER ProducTs (INTERACT) meeting,¹⁰ and as detailed here, in pre-investigational new drug (IND) and Type C meetings, and obtained the drug development incentives Orphan Drug Designation (ODD) and Rare Pediatric Disease Designation (RPDD) from the FDA.¹¹

To test an investigational product in humans, a sponsor must, at a minimum, file an IND application with the FDA and obtain a “study may proceed” notification. The pre-IND is a Type B meeting with the FDA that occurs before the submission of an original IND and, compared with an INTERACT meeting, is intended for a more mature stage of translation of the investigational product.¹² A pre-IND meeting is most appropriate when the sponsor has defined the manufacturing process, completed proof-of-concept (POC) pharmacological studies, has an IND-enabling toxicology study plan, and developed a draft clinical study design for the investigational product. In the development of AAV9-hPCCA, we continued the pharmacology, chemistry, manufacturing, and controls (CMC) and clinical protocol design activities, incorporating INTERACT feedback, and then proceeded to a pre-IND meeting. The FDA reviewed the overall preclinical development plans for AAV9-hPCCA at this meeting and responded to our specific Pharmacology/Toxicology, CMC, and clinical questions.

In addition to a pre-IND meeting, a sponsor can have other formal regulatory meetings with the FDA, such as a Type C or Type D meeting for early consultation on targeted topics.¹² In contrast to a pre-IND meeting, multiple Type C or Type D (more narrowly focused) meetings can be requested by the sponsor to gain clarification on post-pre-IND development questions that arise, e.g., preclinical, clinical, and manufacturing. After the pre-IND meeting, we continued the development of AAV9-hPCCA and subsequently obtained valuable clarifications from the FDA on surrogate biomarker endpoints and CMC issues through a Type C meeting.

The goal of this article is to share regulatory lessons learned during the pre-IND and Type C meetings for AAV9-hPCCA, which can be applied to other AAV gene therapy translational programs. Here, we have summarized the process, our questions, and the FDA’s feedback for each of the two regulatory meetings. The meeting requests (MRs), meeting packages (MPs), preliminary responses, and FDA meeting feedback are provided on the PaVe-GT website (https://pave-gt.ncats.nih.gov/).¹³

PRE-IND MEETING FOR AAV9-hPCCA

Strategy

After the INTERACT meeting in July 2021,¹⁰ we applied the FDA’s feedback and recommendations to continue development of the investigational product AAV9-hPCCA for PCCA-related PA. We focused on the following IND-enabling activities: (1)

Pharmacology: POC studies using the research-grade or early process development AAV9-hPCCA batches were completed, and an additional non-Good Laboratory Practices (GLP) efficacy and safety study in the PA animal model was planned;

(2)

CMC: a pilot-scale (50 L) lot of AAV9-hPCCA was manufactured to define the manufacturing process and release specifications for the product;

(3)

Toxicology: the GLP toxicology study protocol was drafted;

(4)

Clinical: the clinical protocol synopsis draft for the first-in-human (FIH) Phase 1/2 study was updated based on INTERACT feedback.

Upon completion of these activities, the PaVe-GT drug development team worked with a regulatory Contract Research Organization (CRO) to review all available AAV9-hPCCA information and study results and to strategize the next steps in product translation. The PaVe-GT team included more than 20 scientists with expertise in genomics, AAV vector design, disease animal models, biologics manufacturing, assay development, and Pharmacology/Toxicology studies, clinicians/geneticists specializing in organic acidemias natural history studies (NHS), and project managers (PMs) experienced in translational research and regulatory strategy. The team determined that the FDA’s feedback was needed on Pharmacology/Toxicology, CMC, and clinical issues, especially in the context of rare diseases and creating platform efficiencies. Specifically, we needed FDA’s feedback on: a) our proposed additional non-GLP efficacy and safety protocol, which was a Pharmacology/Toxicology study planned to be performed in the same PA mouse model used in the POC studies and neonate wild-type (WT) controls, and a GLP toxicology study in adult WT mice; b) our proposed manufacturing process for AAV9-hPCCA, product quality attributes, and stability testing plans; and c) the clinical protocol synopsis for testing AAV9-hPCCA in children and adults with PCCA-related PA. Overall, regulatory strategy and the details on goals, readiness, and process are shown in Figure 1.

Figure 1.

AAV9-hPCCA regulatory strategy, with a focus on the pre-IND meeting goal, readiness, and package content. The regulatory strategy for the first product of PaVe-GT, AAV9-hPCCA, included INTERACT, pre-IND, and Type C meetings prior to IND filing and executing the FIH clinical trial. The pre-IND meeting goal, readiness, and meeting package content are summarized, along with highlights of the pre-IND meeting logistics, process, and timeline. AAV, adeno-associated virus; AAV9-hPCCA, AAV serotype 9 human propionyl-CoA carboxylase alpha subunit; IND, investigational new drug; INTERACT, INitial Targeted Engagement for Regulatory Advice on CBER/CDER ProducTs; MR, Meeting Requests; MP, meeting package; PaVe-GT, Platform Vector-Gene Therapy.

Meeting and materials preparation

A small team led by the PMs kicked off the pre-IND meeting preparation by collecting and organizing the documentation on the current AAV9-hPCCA development, followed by data evaluation to determine readiness for the meeting. We then drafted the questions for the FDA based on the needed feedback and created a target schedule for pre-IND–related activities, including MR and MP preparation. Milestones of the pre-IND meeting and documentation preparation process, as they integrate with the AAV9-hPCCA development process, are shown in Figure 2A.

Figure 2.

Overview of the pre-IND meeting for AAV9-hPCCA. (A) The process for meeting preparation includes key details on planning, documentation, and the meeting itself. (B) The pre-IND meeting timeline for AAV9-hPCCA includes milestones and time periods for all steps of the process.

To work in parallel on the MR and MP, the PM team was crucial in leading the overall pre-IND preparation process, setting up digital communications, ensuring efficient information sharing across subteams, and organizing all documentation using a team collaboration platform. In the MR for AAV9-hPCCA, we included a brief description of the product, the proposed indication, and planned Pharmacology/Toxicology, CMC, and clinical questions for the FDA. This request (∼10–15 pages) helped the FDA assemble reviewers with subject matter expertise in AAV gene therapies and rare metabolic diseases. Approximately 30 days after the FDA received our MR, they responded, informing us that a meeting (teleconference) was scheduled. The MP prepared was an extensive document (∼300 pages in length), which included comprehensive Pharmacology/Toxicology, CMC, and clinical information (see description in Fig. 1 and summary of MP preparation in Table 1). The MP was submitted on time via the Electronic Submission Gateway to meet the FDA’s deadline of at least 30 days prior to the pre-IND meeting date (see timeline of pre-IND activities in Fig. 2B).

Table 1.

Key Highlights of the Pre-IND MP Preparation for AAV9-hPCCA

MP component by subject	Preparation steps and information included
Pharmacology/Toxicology
POC Study Reports	• Collected, analyzed, and organized relevant POC data from different studies. • Used a study report template to fill in background information, study plans, materials and methods, results (Figures and Tables), and conclusions. • Included a description of the PCCA-related PA mouse model, the AAV9-hPCCA lead candidate, and in vitro and in vivo experiments in which AAV9-hPCCA was tested. Note: Peer-reviewed publications can replace the study reports if available.
Non-GLP efficacy and safety study plan	• Drafted an efficacy and safety study protocol in the PA mouse model (neonates) with WT age-matched controls, following a prospective study design. • The additional safety data from this study would complement the GLP toxicology study in WT adult mice.
GLP toxicology study plan	• Planned a 6-month study in adult WT mice to be carried out at a CRO. • The study was designed using our toxicology team’s and CRO’s expertise with AAV gene therapy studies, published guidance from the FDA and ICH, and the FDA’s comments at the INTERACT meeting.
CMC	• Assembled and organized the manufacturing process and product quality information. • Included: (1) Description of the proposed GMP manufacturing process (based on a pilot-scale 50-L lot of AAV9-hPCCA). (2) List of proposed DS and DP specifications based on the 50-L lot release testing results. (3) Accelerated and real-time stability testing plans for the DP. (4) Device compatibility study protocol. Note: Data were provided by the CMO and organized by the regulatory CRO. • Strategized the regulatory approach for AAV9-hPCCA in the context of the PaVe-GT platform, because the experience with AAV9-hPCCA would also inform the development of the next three products in our program, especially regarding the quality of raw materials (starting cell line, cell banks, and plasmid cell banks) and critical quality attributes.
Clinical
Clinical protocol synopsis	• Updated the clinical synopsis for the FIH Phase 1/2 study to incorporate FDA feedback from the INTERACT meeting. • Implemented additional changes based on the latest POC study data and new literature (clinical studies related to AAV9 gene therapies for rare diseases, mRNA experimental therapy for PCCA-related PA, and immunosuppressive regimens used in association with AAV gene therapy). • Included proposed surrogate biomarkers and a related primary efficacy endpoint.
NHS protocol	• Included the protocol for the ongoing NHS, together with biomarker data collected from this study, which support further evaluation in the Phase 1/2 clinical trial.
Other
INTERACT response matrix	• Included responses to the FDA’s comments, as captured in their INTERACT meeting minutes.
Supporting references	• Added the most relevant publications to this pre-IND application.
Questions	• Formulated questions with relevant background information and supporting data.

CMC, chemistry, manufacturing, and controls; CMO, Contract Manufacturing Organization; CRO, contract research organization; DP, drug product; DS, drug substance; FDA, Food and Drug Administration; FIH, first-in-human; GLP, good laboratory practices; ICH, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; IND, investigational new drug; INTERACT, INitial Targeted Engagement for Regulatory Advice on CBER/CDER ProducTs; MP, meeting package; NHS, natural history study; PA, propionic acidemia; PaVe-GT, Platform Vector-Gene Therapy; PCCA, propionyl-CoA carboxylase alpha subunit; POC, proof-of-concept; WT, wild-type.

Meeting with the FDA

Three days prior to the meeting date, the Agency provided us with preliminary responses to our questions and a sample agenda for the pre-IND meeting. In their responses, the FDA reviewers, who included more than 10 experts in viral vectors, immunology, Pharmacology/Toxicology, and CMC/manufacturing of gene therapies, and clinicians, provided valuable feedback for some aspects of AAV9-hPCCA development not targeted by us in the questions to the Agency. Within hours of receiving these responses, the team reviewed all the feedback and prioritized the top questions for discussion at the meeting. In addition, we identified preliminary responses that could benefit from added clarification from the FDA. For these items, our subject matter experts (SMEs) prepared a slide presentation to present at the actual meeting, ensuring there were no new data or findings that had not been previously included in the MP. Our slide presentation and proposed meeting agenda were then submitted to the FDA at least 24 h prior to the meeting.

The SMEs, two patient advocates, and the PM team participated in the meeting with the FDA. In their opening remarks, the patient advocates shared their experience living with family members with PA and urged the FDA to expedite new therapies for this devastating rare disease. The meeting discussions covered: (1) the suitability of the PA mouse model to support the proposed clinical trial assessments; (2) CMC/drug product (DP) potency assay; (3) residual host cell DNA (rHCD); d) the planned use of data from the proposed non-GLP efficacy and safety study and the GLP toxicology study to inform clinical trial dosing; and (4) the target population for the Phase 1/2 clinical trial (details of preliminary FDA responses and meeting discussions organized into Pharmacology/Toxicology, CMC, and clinical feedback are captured in Table 2A–C). While the pre-IND meeting cannot be recorded, according to FDA rules, we captured meeting minutes that we later compared with the official minutes received from the Agency to ensure sponsor and Agency alignment on all points discussed. The discussions with the FDA reviewers were helpful and led to the clarification of issues raised in the MP and preliminary responses (see highlights in Fig. 3A). The FDA’s position was supportive of the development of AAV9-hPCCA with a line of sight toward longer-term regulatory strategy and the possibility of leveraging the Phase 1/2 study data to support a license application.

Table 2.

Summary of the FDA Feedback and Recommendations from Both the Preliminary Responses and Teleconference Discussions, as Provided in the Final Meeting Minutes

Sponsor question and supporting data for CMC	Summarized FDA feedback
CMC information
Question:Does the Agency agree with the release specifications for the drug substance and drug product? Supporting information from the sponsor: • Description of the manufacturing process and process controls using data from a pilot-scale 50-L lot. • Certificate of testing for the 50-L lot (DS and DP). • Proposed 200-L manufacturing process and testing plan, including potency; this is the process that will be used to manufacture material (DS/DP) for the GLP tox study and the FIH clinical trial. • Other supporting information: raw materials used and testing facilities.	CMC strategy: • If this rare disease gene therapy is to support a marketing application, the CMC information should be appropriate for late-stage clinical development. Potency testing plan: • Qualitative assessment of transgene expression (Western blot) as a potency assay may be acceptable for an early-phase clinical trial. To utilize data from the proposed trial for a marketing application, a quantitative, accurate, and precise potency assay should be developed early in clinical development. Suggested potency approaches: (a) A quantitative assay reflecting the product’s mechanism of action (e.g., enzymatic assay) or (b) A precise next-generation sequencing (NGS) assay (capable of quantifying vector genomes with mutations in the transgene sequence) to ensure that the DS sequence is unaltered, used in conjunction with quantitative measurement of transgene expression (mRNA or protein), may be adequate to ensure product potency. Release criteria: • In early development, release acceptance criteria for impurities and residuals may be set relatively broad, given the limited experience, but can be tightened based on appropriate acceptance criteria experience gained from manufacturing preclinical, engineering, and clinical lots. • Residual host cell DNA (rHCD) in the 50-L drug product is higher than the World Health Organization-recommended limit of 10 ng/dose. The FDA recommended justifying the proposed limit(s), providing a comprehensive risk assessment, and discussing process optimization plans to reduce the level of rHCD in the commercial product when submitting an IND. • In the IND, the sponsor should provide a detailed description of the release assays (procedures, controls, sensitivity, etc.), assay qualification status, and a qualification report, if available, for each cGMP drug product lot. • In the IND, provide a description of each non-compendial test performed for qualification of the master cell bank, including the assay procedure, controls and standards, and sensitivity, as applicable.
Question: Does the Agency agree with the proposed storage, preparation, and stability testing plan?Supporting information from the sponsor: • Storage conditions and container closure for the drug product after release • Accelerated and real-time stability testing plans	Overall, the proposed plan was deemed acceptable with a few recommendations:DP stability plan: • Include a Western blot assay for PCCA expression. • Develop a quantitative potency assay and include it in product development as soon as possible. • In lieu of sterility testing between t = 0 and the last timepoint of the stability testing plan, consider performing a container closure integrity test. DP storage, preparation, and infusion formulation procedures: • Provide a description of established shipping procedures, product stability during DP shipping, and procedures to ensure chain of custody, product quality, and stability. • Product storage, shipping, handling, preparation, and administration procedures should be supported by stability and compatibility study results. • If using a non-compendial diluent to formulate the final drug dose, describe the manufacturing and release testing plan, as well as the stability testing plan.
Question: Does the Agency agree with the plan for assessing the compatibility of the drug product with the preclinical and clinical administration devices?Supporting information from the sponsor: • Proposed detailed compatibility (in-use) study plans for preclinical and clinical infusion devices	Overall, the proposed plan was deemed reasonable with a few recommendations:Pharmacy: • Provide the pharmacy manual in the IND. Compatibility study: • Specify hold times for diluted DP in the syringe prior to holding it for 0, 30, 60, 90, and 120 min in the administration device and indicate hold temperature and duration for diluted DP in the syringe before being held in the administration device; hold conditions of the diluted DP in the syringe should represent a worst-case condition, as described in the pharmacy manual. • The diluent used in the device study should be the same as that used to formulate the DP in the pharmacy.
	Recommended FDA Guidance for Industry: “Container and Closure System Integrity Testing in Lieu of Sterility Testing as a Component of the Stability Protocol for Sterile Products” (February 2008).¹⁴
Pharmacology/Toxicology
Questions:Does the Agency agree that the mid-dose in the GLP toxicity study or the highest tested dose, whichever is found to be the No Observed Adverse Effect Level, supports the proposed starting and maximum doses for the clinical trial?Does the Agency agree that the combination of data collected from the 6-month GLP toxicology study using slow bolus tail vein injection in adult mice and the toxicity data from the non-GLP efficacy study using facial vein injection in mouse pups, along with the proposed clinical age de-escalation, support the minimum age of clinical study participants?Does the Agency concur that, pending acceptable results in the planned preclinical studies, the proposed preclinical development program is IND-enabling for a clinical study in patients with a PCCA-related PA, starting with adolescent or pediatric patients and then opening enrollment to eligible patients older than 3 years?Supporting information from the sponsor: • Completed POC study report(s) • Clinical protocol synopsis for AAV9-hPCCA testing in an FIH study • Study plans/protocols including proposed dose groups, mode of administration, number of mice, and assays/tests to be performed: a) Protocol of the planned non-GLP efficacy study in a PCCA-related PA mouse model b) Protocol of the planned GLP toxicology study plan in adult wild-type mice	Overall, the FDA found that sponsor-provided data were insufficient for the FDA to agree with the proposed dose levels in the FIH clinical trial.The FDA reviewed our protocols and provided detailed guidance and recommendations for both non-GLP and GLP Pharmacology/Toxicology studies, in terms of animal model selection, endpoints, biodistribution, and study performance, as summarized below:Proposed non-GLP safety and efficacy study: In addition to what we proposed in the pre-IND, the FDA asked to include the following parameters or justification: • Initially, the FDA suggested (in the preliminary written responses) exploring the use of a less severe PA mouse model to allow the assessment of all parameters since the current neonatal model made measurement of several endpoints challenging due to early deaths among control (untreated knock-out mice). However, after discussion during the teleconference, the FDA tentatively agreed with conducting the proposed non-GLP study in the originally selected PA mouse model based on its ability to recapitulate the features of severe pediatric PA and the use of concurrent untreated WT controls to aid with data interpretation. • The FDA asked for justification of the selection of the PCCA-/- mouse model in the IND (comprehensive discussion and supporting data about the biological relevance of the mouse model to the target population in terms of life span, disease progression, etc.) and to discuss the dose response relationship of AAV9-hPCCA and the levels of improvement in endpoints observed in the non-GLP study. • Confirm the genotype of all animals. • Include daily cage-side observations for morbidity and clinical signs. • Measure plasma ammonia levels. • Assess enzyme activity and protein expression of hPCCA in the brain and kidney • Assess 2-MC in the liver, kidney, heart, and brain. • Perform histopathology of heart, liver, kidney, and brain in all animals. • For any unscheduled deaths, perform comprehensive clinical pathology, gross and histopathology on a complete list of tissues to determine the potential cause of death. • Increase the number of animals to ensure collection of an adequate volume of blood for all protocol-specified parameters. Proposed GLP toxicology study: In addition to what we proposed in the pre-IND, the FDA asked to include the following parameters or justification: • The FDA tentatively agreed with the sponsor’s rationale for conducting the GLP toxicology study in adult WT mice after discussions regarding (a) technical challenges associated with neonatal mice (e.g., injection procedure, limited blood volumes for toxicity endpoints) and (b) both WT adult and neonatal mice are being used for safety evaluation across the proposed pivotal POC and toxicity studies to collect robust toxicology data. • Add rationale in the IND for use of WT animals, including dose justification (biological activity based on POC studies and bracket proposed clinical doses). • Include the rationale for proposed dose levels and sacrifice timepoints (should coincide with onset, peak, and plateau of PCCA transgene expression), as well as the scoring system used for FOB assessment. • Ensure that qualified personnel perform the assessment and that data analysis is masked to control and experimental study groups. • Collect and archive tissues from all animals for biodistribution evaluations, but initial analysis can be limited to the control and the high-dose groups for major PA-affected tissues. • Determine PCCA transgene expression in tissues that are positive for vector DNA, and the quantitative PCR assay used to evaluate biodistribution should be qualified. • For histopathology assessment, include any gross lesions and the dorsal root ganglia. • For any unscheduled deaths, perform comprehensive clinical pathology, gross and histopathology, on a complete list of tissues and other analyses to determine the potential cause of death. • Provide documentation that the study was conducted in compliance with GLP or a brief statement of the reason for noncompliance; standardized datasets in the SEND format may be required. Other comments applicable to all non-clinical studies: • Materials used: ◦ Provide a tabulated summary of similarities and differences between various nonclinical and clinical lots of AAV9-hPCCA used in the studies (manufacturing process, vector genome titer, infectious titer, full:empty ratio, final product formulation, etc.). ◦ Use the same assay for vector titer determination in non-clinical and clinical studies. ◦ Retain material from all lots. • Study performance: ◦ Avoid potential bias in the studies–randomize assignment of animals to study groups, perform appropriate staggered dosing. ◦ Ensure masked assessment of study parameters and that qualified personnel perform the study and analyze the data. ◦ For dosing calculations, use the proposed dosing unit (vector genomes/kg) as this will be the unit used for clinical dosing. ◦ Document any dosing errors in raw data and final study report. ◦ Explore opportunities for reducing, refining, and replacing animal use. ◦ Strongly recommend oversight of the conduct of all non-GLP toxicology studies and final study reports by an independent quality assurance unit/person. • Confirm compatibility of nonclinical lots of AAV9-hPCCA with nonclinical delivery system. • Provide a comprehensive discussion of how animal studies inform the risk of complement activation and subsequent potential thrombotic microangiopathy following administration of AAV9-hPCCA in patients with PA, who may have compromised liver and cardiac function. • Provide complete study reports for all non-clinical studies used to support the safety and rationale of the proposed clinical trial. • Include copies of publications directly supportive of the proposed clinical trial and provide a comprehensive discussion of each cited publication to explain how the data support the clinical trial.
	Recommended FDA Guidance for Industry & other resources: • “Study Data Technical Conformance Guide”¹⁵ • E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1)-Guidance for Industry “ (March 2018)¹⁶ • Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products” (November 2013)¹⁷ • OTAT Learn Webinar Series (updated to OTP Learn)¹⁸
Clinical
Question: Does the Agency agree with the proposed FIH study design, including participant inclusion/exclusion criteria, dosing rationale, study population rationale, staggering of IP administration, stopping rules, safety oversight, and safety and efficacy endpoints?Supporting information from the sponsor: • Natural history study protocol • Clinical trial synopsis for open-label, externally controlled study to assess the safety and preliminary efficacy of two doses of AAV9-hPCCA in children with PCCA-related PA (eligibility criteria, dose groups, study objectives, and endpoints, etc.)	The FDA did not agree with the proposed plan based on the information provided in the pre-IND package and made the following recommendations:Study design: • The FDA agreed with conducting dose exploration but indicated that the sponsor provided insufficient preclinical data to support the specific proposed doses in the FIH study. • Study design was deemed acceptable for an early-phase study, but the FDA recommended that the IND sponsor maximize the knowledge to be gained from the study (e.g., interpretability of data from each subject enrolled). • The FDA expressed concern that the proposed comparator arm would limit the interpretability of efficacy data generated from this study; the agency instead suggested conducting a larger randomized controlled trial or at least including a concurrent control group. Study population: • The proposed study plan included only children since adult patients with PA would be less likely to derive clinical benefit (they may already have accumulated potentially irreversible organ damage). The plan also stated that the dosing would start in adolescent patients, if available. The FDA suggested obtaining preliminary safety and tolerability data in adults for the FIH study and potentially including adults who may not have reached end-stage disease, given the wide phenotypic spectrum of PA. • The FDA suggested exploring the possibility of initiating the study in adults with a favorable benefit/risk profile; however, adults with intermediate forms of PA have a good quality of life, and inclusion into the trial would not represent a favorable benefit/risk profile. The FDA asked to include the rationale and evidence to support the proposed plan in the IND. • To enroll children when the clinical investigation is associated with more than a minor increase over minimal risk, these risks must be justified by the anticipated direct clinical benefit for each child (21 CFR 50.52) and the prospect of direct benefit must be evidence-based from clinical and animal studies. Eligibility criteria for inclusion and exclusion: • Provide a rationale for the specified minimum weight of 15 kg. • Provide thresholds for the biomarkers (changes in metabolite levels) that will be used to determine eligibility and consistency in enrollment. • To ensure a comprehensive FDA review of the benefit/risk profile for patients diagnosed utilizing a molecular gene analysis, include detailed information regarding the genetic testing (lab information and Clinical Laboratory Improvement Amendments; capability, detailed method and validation status, quality controls and historic information from the lab). • Limit the initial population in this FIH study to subjects who are unresponsive to optimal standard-of-care therapy or justify a broader FIH study population. • Provide a justification for the neutralizing antibody titer threshold for eligibility. • To ensure subject safety, do not administer AAV9-hPCCA within 4 weeks after an illness (febrile or nonfebrile) and refrain from administering the product within 4 weeks of a vaccination. • Provide the laboratory parameter range for international normalized ratio and partial thromboplastin time required for inclusion of subjects. Dosing rationale: • Provide data from nonclinical studies in the IND to justify the proposed dose levels for the different cohorts. • To guide dose escalation or expansion, provide objective criteria and clarify the acceptable safety profile. • Since the biomarkers (metabolite changes) have not been validated for PA, consider using maximum tolerated dose to make dosing decisions when moving into Phase 2/3 studies. Safety endpoints: • Proposed safety endpoints were deemed reasonable: incidence of treatment-related adverse events, treatment-emergent adverse events, serious adverse events and their relationship to AAV9-hPCCA administration based on CTCAE v5.0. Efficacy endpoints: • Some endpoints may be impacted by confounding variables and make study data uninterpretable; and, hence, the FDA advised the sponsor to justify the proposed efficacy endpoints (e.g., annualized frequency of hospitalization/ER visits, total days spent in the hospital/year, and severity of metabolic acidosis and/or hyperammonemia) • Trial protocol should clearly define the dietary goals and management; consider a run-in period for optimizing and stabilizing the patient’s diet and ensure selection of compliant patients. • Growth may be challenging to assess, hence, clinically meaningful outcomes should be chosen that can be reliably interpreted. • For all clinical outcome assessments supporting regulatory decision-making and labeling claims, provide detailed information (instrument framework, evidence of content validity, proposed scoring algorithms and thresholds, plans and results from evaluation, and handling missing data) for FDA review prior to study initiation. • Consider caregiver- and patient-reported outcomes as exploratory endpoints. Staggering: • Provide a data-driven rationale and justification for the proposed intra-cohort staggering interval of 12 weeks. • Stagger at least the first three subjects in each dose cohort. • Specify an inter-cohort staggering interval and a data-driven justification. Stopping rules: • The FDA did not agree with the proposed rules for suspending the study as they are only triggered if attribution is made to the product, its administration, or other study-related procedures. • Preclinical data are needed for the FDA to determine if stopping rules are adequate. The FDA suggested updating the rules based on nonclinical studies and safety data from clinical studies of related products. • If a stopping rule is triggered, enrollment and treatment of new subjects in the study should be paused, and an independent data monitoring committee should evaluate the data, decide causality, and advise on how to proceed. • The FDA did not agree that death or malignancy attributed to the product should be limited to 1 year to qualify as a study stopping rule and asked the sponsor to remove any time bounds for serious safety signals attributed to the product. Safety monitoring plans: • Include a peri-infusion monitoring plan (see details in redacted document on PaVe-GT website) and specific discharge instructions. • Provide defined infusion (starting, slowing, and stopping) rules and specific criteria, including vital ranges that would trigger these decisions. • Provide specific discharge criteria, including vital signs and clinical status; include detailed discharge instructions for subjects.
Question: Does the Agency agree with the plan to use historical and concurrent data from the natural history study as a comparator arm for the FIH clinical trial?Supporting information from the sponsor: • Natural history study protocol • Clinical trial synopsis including eligibility criteria, dose groups, study objectives, and endpoints, etc.	While the FDA found the plan acceptable for an early-phase study primarily focused on assessing safety and tolerability, the agency emphasized the need for an appropriate external comparator arm for determining efficacy. Extensive discussions were held, as summarized below: • The FDA emphasized that the assessments in the study and comparator groups must be highly similar for all relevant variables, such as the types, methodology, and schedule, to avoid confounding comparisons on efficacy and safety outcomes and limit data interpretability. The FDA also indicated that a potential selection bias may occur when research participants are knowledgeable of the treatment. • The FDA pointed out the differences in the ongoing NHS and planned clinical trial design, such as baseline characteristics, dietary management, concomitant therapies, assessments, methodology, and schedule of assessments. • While the FDA reiterated its recommendation of a double-blind, randomized, placebo-controlled trial with a control arm receiving only optimal standard of care, the agency acknowledged the challenges of conducting such a trial and suggested the option of using an external control group where data are generated prospectively to minimize selection bias and to ensure comparability between the external control and FIH study. • During the teleconference discussion, the challenges of enrolling into a placebo arm because of the disease rarity and the invasive nature of the proposed gene therapy protocol utilizing a PICC line were discussed. Accordingly, the plan to use the NHS as a comparator study was discussed extensively: ◦ NHS participants already receive a standard workup to which additional evaluations may be added if clinically indicated. ◦ The same group of investigators who run the NHS will be involved with the performance of assessments during the Phase 1/2 trial. ◦ The FDA emphasized that the external control group and Phase 1/2 group should be similar in baseline characteristics, management of diet and concomitant therapies, as well as the types, methodology, and schedule of assessments. ◦ The sponsor stated that there may need to be differences in the timing of the assessments between the NHS cohort and the Phase 1/2 cohort and will provide a rationale and justification for interpretability of each such assessment in the IND.
Question: Does the Agency agree with the proposed safety endpoint, and use of the described surrogate endpoints as the primary efficacy endpoint for the FIH study?Supporting information from the sponsor: • Clinical trial synopsis including eligibility criteria, dose groups, study objectives, and endpoints, etc.	The FDA did not agree with the use of a surrogate endpoint for primary efficacy evaluation and asked for a rationale and data-driven justification for the use of POBT and metabolite changes as surrogate biomarkers that are reasonably likely to predict clinical benefit and why a study based on a clinical endpoint is not feasible. The agency also suggested characterizing the threshold for change in metabolite and POBT needed to show a clinically meaningful effect, demonstrate consistency of response under various conditions, and reliability of metabolite and POBT for quantifying changes in the clinical outcome before and after treatment. Additional recommendations for the sponsor included: • Discussing the quantitative relationship between changes in biomarkers and clinically meaningful endpoints. • Describing the reliability and consistency of biomarker measurements under varying circumstances. • Studying the biomarkers as exploratory pharmacodynamic indicators. • Exploring clinically meaningful endpoints throughout the developmental program • Considering and incorporating patient experience data in the developmental program and determination of clinically meaningful endpoints. • Addressing whether the expected clinical treatment effect size can be demonstrated in a feasible confirmatory trial given the size of the patient population. • Including a mitigation strategy to ensure that gut bacterial propionate production does not interfere with the interpretation of POBT results. • Providing a rationale and supportive evidence in the IND regarding use of Exalenz Breath ID, currently a 510 K-cleared device for H. pylori diagnosis and monitoring, for POBT measurement.
Question: Does the Agency agree with the proposed immunosuppressive regimen?Supporting information from the sponsor: • Clinical trial synopsis including proposed immunosuppressive regimen.	The FDA did not agree with the proposed plan based on the information provided in the package and made the following suggestions: • To ensure subject safety in the event of metabolic decompensation triggered by corticosteroid administration in a subject with hyperammonemia, a characteristic feature of organic acidemias, particularly PA, the FDA recommended to have a risk mitigation plan and increasing the frequency with which ammonia levels are assessed (proposed protocol suggested only once). • Because of the risk of adrenal insufficiency following tapering of steroids below physiological levels after prolonged high-dose corticosteroids, the sponsor was asked to address in the IND: (1) ways to assess and manage suppression of the hypothalamic–pituitary–adrenal axis to avoid adrenal insufficiency and (2) discuss when children will be referred to endocrinology for further assessment, management, and counseling • Discussing the prolonged corticosteroid therapy and other risks with the subjects enrolled in the study.
	Recommended FDA guidance for industry and other resources: • “Long Term Follow-Up After Administration of Human Gene Therapy Products: Guidance for Industry (for further discussion regarding tissue collection and the quantitative PCR assay methodology, including the current CBER/OTP standard for PCR)” (July 2023).¹⁹ • “Inborn Errors of Metabolism that Use Dietary Management: Considerations for Optimizing and Standardizing Diet in Clinical Trials for Drug Product Development” (July 2023).²⁰ • “Considerations for Discussion of a New Surrogate Endpoint(s) at the Type C PDUFA Meeting Request”²¹ • “Patient-Focused Drug Development: Collecting Comprehensive and Representative Input” (June 2020)²²

CBER, Center for Biologics Evaluation and Research; CFR, Code of Federal Regulations; cGMP, current good manufacturing practices; CTCAE, Common Terminology Criteria for Adverse Events; FOB, functional observational battery; ICH, International Conference on Harmonisation; IP, investigational product; MC, methylcitrate; NGS, next-generation sequencing; NHS, natural history study; OTAT, Office of Tissues and Advanced Therapies; OTP, Office of Therapeutic Products; PCR, polymerase chain reaction; PDUFA, Prescription Drug User Fee Act; PICC, peripherally inserted central catheter; POBT, 1-¹³C-propionate oxidation breath test; rHCD, residual host cell DNA; SEND, Standard for the Exchange of Nonclinical Data; WHO, World Health Organization.

Figure 3.

FDA highlights provided during early AAV9-hPCCA regulatory review. (A) Pre-IND meeting key feedback that impacts the IND-enabling studies carried out prior to IND filing; (B) Type C meeting feedback that clarified CMC and clinical questions. CMC, chemistry, manufacturing, and controls; FDA, Food and Drug Administration.

Meeting minutes and post-pre-IND activities

Thirty days after the pre-IND meeting, we received the official meeting minutes from the FDA, which also included a list of templated information applicable to the Pharmacology/Toxicology and clinical sections, with generic regulatory advice and references to applicable FDA guidance (captured in Table 2A–C). In comparing our minutes with the FDA’s minutes, we identified an area related to the target population for clinical testing that required an amendment. We informed the FDA about our understanding of the final discussions regarding the first patient enrolled in each clinical trial cohort, which differed from what was captured in the Agency’s minutes. While this is not a compulsory action, our goal was to ensure that the clinical enrollment aspect was clear, as it has an impact on the performance of the clinical trial. The meeting minutes will guide the rest of the IND-enabling studies for AAV9-hPCCA, the regulatory strategy beyond the Phase 1/2 clinical trial, and the development of the other AAV gene therapies in the PaVe-GT platform.

TYPE C MEETING FOR AAV9-HPCCA

IND-enabling activities undertaken after the pre-IND meeting and continuing review of the recommendations made by the FDA led us to uncover CMC and clinical topics for which we needed further input from the Agency prior to IND filing. Type C meetings enable sponsors to have early consultation with the Agency on a few specific topics related to product review or development. Thus, we decided to engage the FDA in a Type C meeting as part of our regulatory strategy for AAV9-hPCCA.

Strategy

During the pre-IND meeting, the level of rHCD in the 50-L pilot-scale lot of AAV9-hPCCA was determined to be high relative to the World Health Organization (WHO)-recommended limit of 10 ng/dose.²³ Thus, we made adjustments to the manufacturing process to lower the rHCD level in the 200-L engineering lot of AAV9-hPCCA. In addition, we prepared a risk mitigation strategy for addressing the rHCD levels relative to the WHO limit. The assay to evaluate product potency was also discussed at the pre-IND meeting, and based on FDA recommendations, we planned a matrix potency-testing approach that would include the quantification of PCCA mRNA by PCR and of PCCA mutant variants via Next Generation Sequencing (NGS).

After the pre-IND meeting, the clinical team, which included clinical research investigators, geneticists, and nursing staff, continued to collect data on several biomarkers as part of the NHS of PA (NCT02890342) conducted at the NIH Clinical Center.²⁴ Data analysis showed a statistically significant correlation between the 1-¹³C-propionate oxidation breath test (POBT), metabolite levels, and clinical outcome measures. This finding led to proposed changes in the Phase 1/2 study design in which dose escalation would be dictated by safety measurements, with changes in POBT and metabolite levels representing secondary endpoints, not primary endpoints, as initially proposed prior to receiving pre-IND feedback. Their use as surrogate biomarkers for efficacy endpoints in a future clinical trial may be possible based on cumulative data from the natural history and Phase 1/2 studies.

To further de-risk AAV9-hPCCA development, we wanted to obtain FDA’s feedback on the risk assessment and approach to lower rHCD in the DP, the plans for testing and qualifying the planned matrix potency assay for AAV9-hPCCA, and the utilization of changes in POBT and metabolite levels in the Phase 1/2 clinical trial (Fig. 4A). The team decided that this could be done through a Type C meeting with the FDA. After evaluating readiness for the Type C meeting, the PM team led the CMC, clinical, and regulatory subteams to collect, organize, and analyze the data supporting the topics and to formulate the questions for the FDA.

Figure 4.

Overview of the Type C meeting for AAV9-hPCCA. (A) The process for meeting preparation, including the goal, preparation steps, and meeting logistics. (B) The meeting timeline for AAV9-hPCCA, including important dates and preparation periods.

Meeting and materials preparation

Similar to the pre-IND meeting, the Type C meeting was requested by submitting an MR (10 pages), which included background and related questions for the FDA. The regulatory SMEs ensured that adequate data were shared with the FDA to allow the Agency to answer our questions. After the FDA responded to the MR in 21 days by initially granting us written responses only (WRO) (the timeline for all Type C meeting steps is shown in Fig. 4B), we submitted the MP (∼105 pages) 47 days prior to the meeting date and requested conversion of the WRO to a meeting to allow a more definitive dialogue about the surrogate biomarkers. The FDA approved our request.

Preliminary responses and meeting-turned WRO

Five days prior to the meeting date, the FDA provided preliminary responses to our questions. The reviewers agreed with our risk assessment and reporting of rHCD levels for AAV9-hPCCA lots and concurred with our proposed matrix approach to assess product potency using quantification methods for PCCA mRNA expression and PCCA mutant variants. The FDA reiterated its recommendation from the pre-IND meeting to demonstrate the suitability of the NGS method for quantitative analysis of sequence variants (Fig. 3B; details in Table 3) and to set up the assay by spiking mutated sequences in the test sample. With respect to biomarkers, the FDA agreed with our: a) proposed approach to evaluate changes in metabolite levels and POBT in the Phase 1/2 study to assess pharmacodynamic responses to AAV9-hPCCA treatment; and b) updated clinical study design. The FDA offered to cancel the virtual Type C meeting if we found their responses and advice clear and complete, thereby obviating the need for further discussion. We agreed and informed the Agency of our decision to cancel the meeting. However, subsequently, we found it challenging to implement the FDA’s advice to “spike” the test sample with mutant sequences to develop a quantitative NGS assay that can accurately and reproducibly measure the levels of variants in the drug substance (DS) as part of the potency assay. Thus, we submitted an official written request for clarification (two pages) of the spiking method, and the Agency responded in writing within 3 weeks (four pages) restating the need for a spiking study to assess the sensitivity of the NGS assay. We found the communication with the FDA valuable for preparing our IND application for AAV9-hPCCA and planning of the for the remaining three AAV gene therapy products in the PaVe-GT program.

Table 3.

Type C Meeting Questions and FDA Feedback

Sponsor question and supporting data	Summarized FDA feedback
Clinical question:Does the Agency agree with the proposed approach for utilization of the specified surrogate biomarkers for the evaluation of preliminary clinical efficacy (PD response) and associated measures?Supporting information from the sponsor: • Background and responses to biomarker-related FDA feedback from the pre-IND meeting. • Additional biomarker data for POBT and target metabolites and associated rationale for their use in evaluating preliminary clinical efficacy of AAV9-hPCCA	The FDA agreed with evaluating changes in POBT and target metabolite levels in the Phase 1/2 FIH study to assess PD responses to treatment. They found the proposed approach reasonable to evaluate the clinical meaningfulness of changes in those PD biomarkers, including robust data collection during the clinical trial and collection of data from the ongoing NHS.
CMC question: Does the Agency agree with the risk assessment and approach to support the limits of rHCD?Supporting information from the sponsor: • Background and responses to HCD-related FDA feedback from the pre-IND meeting • Summary of manufacturing process improvements to decrease the amount of residual HCD in the AAV9-hPCCA product • Risk assessment including the HCD characterization approach	The FDA agreed with the risk assessment and approach proposed by the sponsor to support the limits of rHCD in the AAV9-hPCCA drug product.
CMC question: Does the Agency agree with the plans for testing and qualifying a potency assay matrix for AAV9-hPCCA?Supporting information from the sponsor: • Background and responses to potency-testing–related FDA feedback from the pre-IND meeting • Proposed matrix approach for potency testing of AAV9-hPCCA (quantitative mRNA in conjunction with NGS testing) • Plan for transgene mRNA expression by quantitative droplet digital PCR • NGS protocol demonstrating method suitability including test example data	Overall, the FDA found the proposed matrix approach to assess product potency for release testing acceptable for initiation of the IND application. The FDA provided additional comments: • The FDA acknowledged the sponsor’s plans to qualify the mRNA assay prior to clinical testing and validate it prior to the start of Phase 2 clinical testing. Accordingly, the FDA requested submission of the SOP and qualification report as part of the initial IND. • Regarding the NGS assay, the FDA raised concern about whether the proposed method was adequate to be qualified for quantitative analysis of vector sequence variants and provided recommendations: • To retain samples from all clinical lots to confirm the sequence purity once the NGS assay is qualified/validated. This may also facilitate future comparability analyses, in case manufacturing changes are introduced. • To ensure that the assay has sufficient accuracy, precision, range, and linearity in quantifying sequence variants over a specified range. • Spiking the test sample with a mutated sequence to assess the sensitivity of the NGS assay. • Upon request for further clarification regarding the spiking study and the sponsor’s request to consider in silico mutants in lieu of a spiking study, the FDA did not agree with this approach. The FDA found that the sponsor’s proposed in silico spiking of variants, with assessment of assay sensitivity only at the level of bioinformatics, was not adequate. • The FDA maintained the recommendation for use of a spiking study to assess the sensitivity of the NGS assay. A quantitative NGS assay that can detect a sequence variant and determine the level of the sequence variant in a product lot is critical to testing potency for lot release. • They recommended that the NGS assay be validated using test samples spiked with AAV vectors with mutations to assess the precision, accuracy, linearity, and range of the assay. • The FDA acknowledged the sponsor’s plans to qualify the NGS assay as part of product development, submit the qualification report after initiation of the IND, and validate the method prior to the start of Phase 2 clinical testing.

After submission of the Type C meeting package, the FDA shared preliminary responses, which the sponsor found satisfactory, and thus, the teleconference was canceled. However, we requested clarification regarding the potency assay from the FDA (refer to Fig. 3B). The following information is a summary of the FDA’s feedback and recommendations from both the preliminary responses and follow-up conversations.

AAV, adeno-associated virus; HCD, host cell DNA; PD, pharmacodynamic.

DISCUSSION

For the first product of the PaVe-GT program, AAV9-hPCCA, we published the experience and materials from the first regulatory interaction with the FDA at the INTERACT meeting¹⁰ and committed to sharing the experience from subsequent FDA interactions, including future IND documentation. By sharing the approach and blueprint of our regulatory experience and application packages, we hope to assist other stakeholders in developing AAV gene therapies for rare diseases. Therefore, we summarize here our pre-IND and Type C meeting experience for AAV9-hPCCA, including goals, process, key feedback, and “dos” and “don’ts” (Fig. 5).

Figure 5.

“Dos” and “Don’ts” to consider in preparing for pre-IND and Type C meetings with the FDA.

At the pre-IND meeting, the FDA reviewers provided advice on the Pharmacology/Toxicology aspects of AAV9-hPCCA development (Fig. 3A). We were initially encouraged to use a less severe disease model of PCCA-related PA in the additional non-GLP safety and efficacy study to enable better assessments of changes in biomarkers compared with untreated PA mice. However, after further discussions, we were able to convince the FDA reviewers that a severe disease mouse model was closer to the clinical scenario, since the risk:benefit ratio did not favor testing of AAV9-hPCCA in patients with mild or intermediate PA. Ideal candidates for participation in our Phase 1/2 study of AAV9-hPCCA are moderate-to-severe patients with PCCA-related PA who do not yet have irreversible organ damage (e.g., cardiac, renal). With respect to Pharmacology/Toxicology studies, the FDA agreed that for our program, safety data from a well-designed GLP toxicology study in WT adult mice and the non-GLP safety and efficacy study in the neonatal lethal PA mouse model could satisfy the requirements for preclinical safety data in support of a FIH Phase 1/2 study.

Regarding CMC aspects of AAV9-hPCCA development, some FDA-provided feedback focused on rHCD content and potency testing. After adapting the CMC process to reduce rHCD and drafting a risk mitigation plan, we shared this information with the FDA at the Type C meeting and received FDA concurrence on our approach. Based on the pre-IND and Type C meeting interactions, we plan to include in the IND: a) a justification for the proposed limit of rHCD content in the DP, which is anticipated to be higher than the WHO-recommended limit of 10 ng per dose,²³ b) a comprehensive rHCD risk assessment plan, and c) plans for manufacturing process optimization to reduce the level of rHCD in the investigational product.

A major lesson learned from the two regulatory meetings with the FDA was that we had to strategize early clinical development with the final goal in mind, i.e., to leverage the proposed Phase 1/2 clinical study data to support a marketing application. Recognizing the regulatory challenges of drug development for very small patient populations, the FDA advised us that CMC information submitted to support the clinical study should be appropriate for late-stage clinical development. This includes the early development of a potency assay applicable up to the submission of a marketing application, collecting quality data to tighten acceptance criteria for quality attributes, and ensuring that product quality data are appropriate for late-stage clinical development. Thus, we plan to develop a quantitative potency assay that best enables lot comparison throughout the lifecycle of AAV9-hPCCA development. The recommendation offered by the FDA at the pre-IND meeting and later clarified at the Type C meeting was to use a matrix approach in which an NGS assay would quantify mutant variants of PCCA in the DS and a quantitative mRNA assay for PCCA in the DP.

Regarding the clinical aspects of AAV9-hPCCA development, we learned from the pre-IND and Type C meetings that our preliminary data on biomarker response (POBT and metabolite levels) from participants in our NHS were insufficient to support their use as surrogate endpoints for efficacy at this early translational stage. However, we hope that ongoing data collection from the NHS and the Phase 1/2 study will support the use of POBT and other metabolite levels as surrogate biomarkers for efficacy in a future clinical trial. Other valuable clinical feedback obtained from these early regulatory interactions included the FDA’s alignment that: (1) a randomized, placebo-controlled clinical trial is not feasible for a very limited patient population such as that of PCCA-related PA and for a high-risk intervention such as gene therapy; instead, the use of an external control for the Phase 1/2 study, based on the NHS, was recommended; (2) the investigational product must be tested first in an adult or adolescent patient before it is tested in children, but if no such adult/adolescent participant is identified, the study can proceed with enrollment of children; and (3) patients with moderate-to-severe disease are the most suitable candidates for enrollment in the Phase 1/2 study. We consider the FDA’s feedback to be specific to the product and based on the sponsor’s submitted data and its unique challenges. Therefore, we ensured that all relevant information and questions on AAV9-hPCCA were included in the meeting packages.

For AAV9-hPCCA, the FDA’s feedback during this early regulatory process (Fig. 3 and Table 2), including feedback related to Pharmacology/Toxicology testing in a single species, product quality improvements, and FIH Phase 1/2 clinical study design, is leading to more efficient development of the other three PaVe-GT candidates and their regulatory strategies. For example, we plan to use a similar manufacturing process, critical quality attributes (with some improvements, as applicable), and an analogous matrix-based potency assay for the next PaVe-GT candidate, AAV9-metabolism of cobalamin associated B. This product will be tested in cblB-type methylmalonic acidemia, an indication similar to PA. Although intellectual property, inventions, and similar protections prevent the free circulation of information among potential gene therapy developers for rare diseases, and gene therapy development is an expensive and high-risk venture, our mission of disseminating PaVe-GT learnings and materials may help overcome some of the scientific and regulatory barriers. The public’s free use of our templates and examples could save resources, educate gene therapy developers, and improve access to information and healthy competition. Upcoming dissemination plans include publication of a PaVe-GT roadmap consisting of the preclinical and regulatory process, ODD, RPDD, and INTERACT templates and examples (already individually published and shared on the PaVe-GT website), as well as the pre-IND and Type C meeting templates for MR and MP, and examples of PaVe-GT regulatory documents. In the future, the PaVe-GT roadmap will also include the IND documentation. To complete the package of PaVe-GT dissemination resources, we plan to publish POC, CMC, and clinical development experiences, and the overall efficiencies from the AAV platform. This effort can also fulfill the broader goal of PaVe-GT to deliver a standardized, platformed, and scalable vector approach that could lead to more efficient AAV-based gene therapies across rare diseases.

AUTHORS’ CONTRIBUTIONS

R.S., R.M.L., and E.A.O. contributed to the conceptualization, methodology, and writing of the initial draft, reviewing, creating figures, and editing the article. The pre-IND and Type C meeting packages and requests used in this article are based on the original research of E.-Y.C., L.L., R.J.C., O.A.S., I.M., C.I.G.-A., J.L.S., and C.P.V., including design and conceptualization of AAV9-hPCCA, creation of the mouse model of PA, demonstration of preclinical efficacy in animal models, clinical research, and biomarker analysis. R.S., R.M.L., O.A.S., I.M., C.I.G.-A., V.M., E.-Y.C., R.J.C., L.L., J.L.S., P.T., X.X., J.D., P.J.B., C.G.B., C.P.V., and E.A.O. contributed to the gene therapy development activities as part of the PaVe-GT team and reviewed and edited the article.

Footnotes

ACKNOWLEDGMENTS

The authors are grateful to the patient advocates from the Organic Acidemia Association and the Propionic Acidemia Foundation for their contribution to the pre-IND meeting. The authors would like to thank Anjali Sarkar for her support in preparing the templates and examples shared on the PaVe-GT website. The authors also thank past and present members of the entire NIH PaVe-GT Team (in alphabetical order): Krishna Balakrishnan, Eggerton Campbell, Catherine Chen, Oksana Dukhanina, Malar Durai, Susan Ferry, Rahul Nandre, Asvelt Nduwumwami, London Toney, Carol Van Ryzin, Sury Vepa, Erik Wagner, and Amy Wang.

DISCLAIMER

This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this article are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.

AUTHOR DISCLOSURE STATEMENT

The authors have no competing interests relevant to this article.

FUNDING INFORMATION

PaVe-GT is funded by the NCATS Cures Acceleration Network and the Intramural Research Programs of NCATS, NHGRI, and NINDS. R.S., R.M.L., V.M., P.T., X.X., and E.A.O. are supported by the NCATS Intramural Research Program (1ZIATR00281). E.-Y.C., R.J.C., O.A.S., L.L., I.M., J.L.S., C.I.G.-A., and C.P.V. were supported by the Intramural Research Program of the NHGRI (1ZIAHG200318-21). C.G.B. is supported by the NINDS Intramural Research Program.

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