Pre-clinical validation of a novel gene therapy for a rare disease in non-human primates: Integrative approach to the CMC scale-up process validation of a gene therapy product for Friedreich Ataxia

Project Information

FRATAXAV

Grant agreement ID: 101114192

DOI

10.3030/101114192

Project closed

EC signature date 5 June 2023

Start date 1 July 2023

End date 30 June 2024

Funded under

• European innovation ecosystems

Total cost

No data

EU contribution

€ 75 000,00

Coordinated by

BIOINTAXIS SL
Spain

Periodic Reporting for period 1 - FRATAXAV (Pre-clinical validation of a novel gene therapy for a rare disease in non-human primates: Integrative approach to the CMC scale-up process validation of a gene therapy product for Friedreich Ataxia)

Reporting period: 2023-07-01 to 2024-06-30

Summary of the context and overall objectives of the project

Friedreich´ ataxia is an autosomal recessive inherited neurodegenerative disease affecting 2-5 individuals for every 100.000 depending on the specific population and as such it is considered a rare disease. However, of all the ataxias, it is the most frequent. Symptoms generally begin between the ages of 5 and 15. The first symptom are usually difficulty walking or gait ataxia due to coordination problems such as clumsiness, movements instability. The ataxia gradually worsens and the symptoms gradually show in the arms and then the trunk. The mutation that causes the disease is found in the frataxin gene encoding a critical protein for the function of the mitochondria, the powerhouse of the body. This mutation, consisting of an expanded GAA code in the intron 1 of the gene, interferes with frataxin expression resulting in decreased protein levels and function. Patients then have low frataxin activity and consequently develop iron metabolism and mitochondria dysfunction which underlies neurodegeneration. Therefore, a protein replacement therapeutic approach is merited in this disease.

Biointaxis S.L. has developed a gene therapy for Friedreich ataxia, BTX-101 (expected commercial name FRATAXAV) to restore frataxin levels in patients. BTX-101 reaches most affected tissues, shows long-term expression, and a good safety profile as seen in pre-clinical research work. For further development into clinical stages, the manufacturing process and quality controls must be clearly defined. Therefore, the objectives of this project aimed to define up-scale manufacturing activities, develop assays to test the potency of different lots, Verified efficacy in a Friedreich ataxia mice mode of a scale-up lotl, and determined the biodistribution and safety profile in Macaca fascicularis. Moreover, regulatory assessment, FTO study, and dissemination activities supports the development of BTX-101 and Biointaxis´ growth and innovation activities.

This project significantly impacts the development of BTX-101 by de-risking the process of scale-up production and the characterization of different lots. Friedreich ataxia at this time counts only with palliative treatments, while gene therapy has the potential for long-term impact on patients´ quality of life.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Objectives and work performed
1. To ensure the CMC batches have the adequate quality for use in animals and eventually for clinical trial, we develop and implement methods to rapidly assess the quality of the BTX-101 looking at at ratio of capsid proteins, viral genome integrity, and deep-sequencing using nanopore technology.
In vitro: A cell culture system was developed to test the potency of BTX-101 by measuring the transduction ratio by dPCR and the transgene protein from each batch
In vivo: The BTX-101 2L was administered to an acute FRDA mice model (Pv-KO) and the motor performance tested and compared with sham FRDA mice and to WT mice.
During this project we also performed a pilot test with one-dose intrathecal administration into 2 NHPs. After three months the tissue was removed and biodistribution of BTX-101 viral genome was determined. Expression of the mRNA was also determined by dPCR. Histological studies showed no findings of toxicology from the BTX-101 administration.

Taken together these QC and potency assays allow us to characterize the BTX-101 batches to evaluate their potential maximum activity, efficacy in FRDA mice and no toxicity in NHPs three months after administration.

2. Data compilation, analysis, and generation of data integration
Data resulting from Nanopore sequencing can yield over 2M reads to be aligned with DNA from the transgene plasmid, RepCap, Ad helper and human genome, making this challenging complex alignment and analysis. The nanopore sequencing analysis has been developed and optimize throughout the project so that we can perform multiple sequence alignments with any potential DNA that may be present during the manufacturing process by generating R-algorithms that facilitate these process

We developed R-algorithms to facilitate the alignment and analysis of DNA sequences of potential DNA contaminants that may be present during the manufacturing process to analyze the results from deep-sequencing by nanopore of BTX-101 from the 2 L batch and previous small batches generated by non-scalable methods.

3. The regulatory assessment of the development program and gap analysis
We have obtained an updated version of the roadmap for product development and have been implementing recommendations from regulatory consultant to best organize data and reporting for regulatory agencies.

4. FTO and new IP including methods for testing activity
An FTO was ordered and is now being expanded to include other elements used during manufacturing to best capture the scope of the long-term flexibility to produce our product.

5. Communication and Dissemination
We have participated in different specialized conferences and as a result are obtaining more visibility to investors and potential partner.

Objectives 3-5 Strengthens Bionitaxis´s competitive position to effectively and efficiently develop the gene therapy product for Friedreich Ataxia, a disease with only palliative treatments, while gene therapy has the potential of long-term impact in patients quality of life.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

1. The results obtained in this project provide strong evidence to support BTX-101 as a gene therapy with clinical potential due to its efficacy in mice models of FRDA, high yield during manufacturing, and a good safety profile according to the pilot NHP results. Access to support at different stages of further development would give the BTX-101 product the impulse to get to later development and clinical trials.

2. Development of potency assays and strategies to de-risk the process of development at early stages is of interest to lower the number of animals studies, and maximize the detection early on of CMC processes that are not efficient.
Innovative approaches to quality control, functional and potency assays will support a more effective and efficient development pathway. Financing this strategies will be of benefit at the mid and long-term to the economy and society.

3. We developed R-algorithms to facilitate the alignment and analysis of DNA sequences of potential DNA contaminants that may be present during the manufacturing process to analyze the results from deep-sequencing by nanopore of BTX-101 from the 2 L batch and previous small batches generated by non-scalable methods. In the era of increasing innovation and artificial intelligence tools, we should be able to implement all tools available to make the manufacturing process and the therapy candidate selection more effective through financing this initiatives.