New Hope for Neuronal Diseases with Polymer Nanoparticle-Based Gene Therapy

Diseases of the central and peripheral nervous system, like Neurofibromatosis type 1 (NF1), are notoriously hard to target for gene therapies. Battelle has developed a new polymer nanoparticle (PNP) delivery system that can deliver to specific cell types, such as neuronal cells. The work, which was recognized as the “Best Poster” at the Controlled Release Society 2024 Annual Meeting in Italy, could pave the way for safer, more effective gene therapies for genetic diseases.

Advancing Gene Therapies for NF1

This work was funded by the Gilbert Family Foundation’s Gene Therapy Initiative and performed in collaboration with research groups at the University of Alabama, Birmingham and the Pennington Biomedical Research Center. Gilbert Family Foundation is the largest private funder for NF1 research in the world, and their mission is to accelerate a cure. NF1 is caused by mutations in the NF1 gene and affects about 1 in 2500 births worldwide. The NF1 gene provides instructions for making neurofibromin, a protein that plays a critical role in controlling cell growth and proliferation. In people with NF1, mutations in the NF1 gene cause a loss of neurofibromin function, which in turn can lead to the formation of tumors along nerves and other symptoms associated with the disorder, such as café au lait spots, learning disabilities and others.

Selumetinib—currently the only US Food and Drug Administration (FDA) approved therapy for NF1—benefits some patients with plexiform tumors but induces toxic side effects and causes tumors to rebound after stopping treatment. Thus, there is an urgent need for new treatment strategies. While a range of genetic therapy approaches have shown potential, two key challenges remain in successfully translating them to the clinic: 1) the development of a universally applicable therapeutic approach for all individuals with NF1 rather than mutation-specific approaches, and 2) the ability to load and deliver large genetic payloads to targeted cells or tissues.

Reconstituting functional neurofibromin in NF1 patient cells presents a promising treatment modality that, due to its wide applicability, has high potential for near-term clinical translation. This can be accomplished by delivering a mutation-free copy of the NF1 gene to cells, which would then express the correct form of the neurofibromin protein. However, this is currently blocked because of the lack of suitable delivery vectors that can encapsulate the large NF1 gene, which at 8.5 kilobase pairs is out of reach of viral vectors such as adeno-associated viruses (AAVs).

Delivering Large Plasmids to Neuronal Cells

For this study, we wanted to identify ways of delivering a full-length human NF1 gene (encoded as a plasmid) into specialized cells in the peripheral nervous system called Schwann cells. Schwann cells are cells that surround and protect the nerves in the peripheral nervous system and have been implicated in the origin of NF1 tumors.

Delivering a large plasmid into neuronal cells presents several challenges. It requires a delivery mechanism capable of carrying a much larger payload than is possible with the current state-of-the-art technologies, such as AAVs or lipid nanoparticles (LNPs), as the NF1 plasmid is four times the size of the largest payload that can be carried by an AAV. Unprotected (bare) genetic material, such as DNA or RNA, is prone to degradation and could also be toxic to cells when delivered alone. A suitable delivery mechanism is needed that stabilizes and protects the genetic payload and minimizes toxicity and potential immune responses.

To overcome these challenges, we used specially designed cationic polymeric nanoparticles (PNP) to encapsulate and deliver the NF1 gene. These nanoparticles:

• Have a very high payload capacity, making them ideal for carrying large nucleic acids. They can also carry other types of materials, such as proteins and small molecules.
• Can be engineered to target specific cell or tissue types.
• Are non-immunogenic (unlikely to trigger an immune response) and can be re-dosed if needed.

Advanced PNP Synthesis with HIT SCAN™

Rational design of PNPs, while promising, is yet to be fully realized due to the complexity of biological systems and the limitations of current modeling techniques. Our approach here was to use Battelle’s High-Throughput Synthesis, Characterization and Assessment of Nanoparticles (HIT SCAN™) platform which enables researchers to synthesize, evaluate and test large quantities of PNPs in a systematic and efficient manner to identify the most optimal “hit” candidates.

In order to achieve desirable chemical and interfacial properties relevant for NF1, we custom designed a chemically diverse library of ~450 unique polymers by combining a set of carefully selected monomers in different ratios. The polymers were synthesized using a RAFT (reversible addition, fragmentation and chain transfer) polymerization process, as this approach allows us to synthesize polymers with controlled molecular weight, low polydispersity and tailored end-group functionality.

Once synthesized, the polymers were self-assembled into cationic PNPs, loaded with the NF1 gene and tested using the HIT SCANTM platform which incorporates iterative rounds of design, synthesis and screening. The process is circular:

• PNPs are synthesized using advanced automated methods, creating a library of likely nanoparticle candidates.
• The candidates are screened through rapid and high-throughput in vitro and in vivo testing methods.
• Data from the screening tests are used by the ML/AI model to learn from and then predict new, optimized PNP compositions, which are synthesized and screened again.

Using this screening platform, we were able to identify a group of chemically distinct PNPs that were uniquely capable of carrying and delivering the large NF1 gene to human Schwann cells in vitro. Further, the identified PNP “hits” were used to demonstrate partial neurofibromin restoration in vitro in human Schwann cells. The efficacy of the best performing PNP “hit” candidate was then confirmed with in vivo testing in rodent models which showed expression of the gene in animal brain regions.

This, to our knowledge, is the first-ever demonstration of delivering the large full length human NF1 gene to neuronal cells in vivo. The high-throughput nature of the platform allowed us to quickly identify the hit PNP, vastly reducing development timelines and enabling rapid synthesis of viable delivery candidates.

One Step Closer to a Cure

More work remains to be done to develop a cure for NF1, but this research brings us one step closer. We were able to demonstrate that it is possible to deliver a large genetic payload into neuronal cells that are highly resistant to transfection. The study provides a basis for future optimization of non-viral gene delivery vehicles for various disease modalities.

PNPs offer significant advantages over viral delivery mechanisms and would make gene therapy more accessible, affordable and effective in the future. In addition to their high payload capacity, PNPs are faster and cheaper to manufacture than AAVs and offer high shelf stability at room temperatures. PNPs are also safer, especially for therapies requiring repeated dosing. Unlike viral vectors, which can trigger strong immune responses and limit the ability to administer repeated doses, PNPs are generally less immunogenic, meaning they are less likely to be recognized and attacked by the immune system. These characteristics would make PNPs a safe and versatile delivery option for a broad range of genetic therapies.

The implications of our research go beyond NF1. There are many genetic disorders and diseases that affect the nervous system, where effective gene delivery has remained a significant challenge. Successfully delivering genetic therapies to these hard-to-reach cells could open new doors for treating a wide range of conditions, from neurodegenerative disorders like ALS and Huntington’s to muscular diseases such as Duchenne muscular dystrophy. Each advancement in targeted, non-viral delivery methods brings us closer to safer, more accessible gene therapies for conditions previously thought untreatable.