Non-viral gene therapy offers hope for chronic low back pain

In a recent study published in the journal Biomaterials, researchers developed a novel non-viral gene therapy to treat discogenic back pain (DBP) by delivering the transcription factor Forkhead Box F1 (FOXF1) using engineered extracellular vesicles (eEVs) into degenerative intervertebral discs (IVDs) in vivo.

Chronic low back pain (LBP) is a growing global problem due to an aging population and worsening opioid problems. Current treatments include short-term relief or expensive surgeries, highlighting the need for non-addictive and less invasive therapies.

Current biological approaches, including growth factor administration, cell therapy, and viral gene therapy, can reduce degeneration in animal and human models. However, issues such as short-term effects, poor long-term efficacy, and unnecessary immunogenicity and oncogenicity may hinder the direct application of these approaches.

In this study, the researchers established a non-viral gene therapy for intervertebral disc degeneration (IVD) using FOXF1-eEV.

The researchers transfected primary mouse embryonic fibroblasts (PMEF) with plasmids containing FOXF1 or pCMV6 as a control and characterized eEV samples using nanoparticle tracking assay (NTA).

They assessed efficient loading of molecular cargo into eEVs using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and conventional PCR. Western blot analysis identified FOXF1 and EV-specific proteins in eEV formations. The team used plasmids that enhance the upper and lower polylinker regions to determine the presence of FOXF1 plasmid DNA in donor cells and generated eEVs.

They examined full-length mRNA produced from plasmid DNA in eEVs and donor cells.

Researchers created extracellular vesicles with transcription factors to restore tissue function and alter pain responses in an animal model of DBP.

They identified EVs for the transport and distribution of FOXF1 into damaged intervertebral discs in a mouse model of discogenic back pain to determine FOXF1 eEV inhibition of intervertebral disc degeneration.

The team combined biomechanical testing of mouse intervertebral discs with imaging, extracellular matrix (ECM) changes, and pain responses assessed after 12 weeks to confirm changes in structure and function, as well as pain, induced by the therapeutic intervention.

Preoperative and postoperative pain assessments included micro-computed tomography (micro-CT), magnetic resonance imaging (MRI), mechanical testing, Alcian blue (AB) and picrosirius red (PSR) staining, dimethylmethylene blue test, and immunohistochemistry (IHC).

The study involved a surgical technique in which researchers administered Buprenorphine ER subcutaneously to mice to control post-operative pain.

The team conducted behavioral assessments before surgery and every two weeks from four to 12 weeks after surgery, using a variety of methods including the open field test, cold plate, tail suspension, and wire suspension.

The open field test assessed the spontaneous activity of mice; cold plate tests measured thermal hyperalgesia; tail suspension tests measured axial pain; and wire suspension tests measured strength.

Twelve weeks after surgery, the team dissected the animals' lumbar spines using femoral nerve and artery tracing to identify intervertebral discs between L4 and L5, L5 and L6, and L6 and S1 IVD. They used the L5/L6 IVD to assess histology and determine glycosaminoglycan (GAG) content.

FOXF1 eEVs significantly reduced pain responses while restoring IVD structure and function, including improved disc height, tissue hydration, proteoglycan content, and mechanical properties.

The study focused on the release of FOXF1-loaded eEVs from primary fibroblasts transfected with the transcription factor FOXF1. Quantitative RT PCR showed a significant increase in FOXF1 mRNA transcript levels and full-length transcribed FOXF1 mRNA levels compared to pCMV6-transfected cells.

FOXF1 eEV therapy can reduce pain responses in a lumbar disc puncture model in mice for up to 12 weeks. Female mice showed longer seizure times in the FOXF1-treated group than in the injured group, which lasted for at least 12 weeks after treatment.

FOXF1 eEV therapy improved IVD tissue hydration and height in injured and degenerative animals in vivo, while maintaining hydration levels and IVD disc T2-weighted image intensity.

However, the team observed a reduction in disc height in injured animals and animals treated with pCMV6 eEV. Mice treated with FOXF1 eEV had no reduction in disc height 12 weeks after treatment. Gender did not affect functional outcomes.

FOXF1 eEVs restored the mechanical function of damaged and degenerated IVDs in vivo. Under axial stress, FOXF1 eEVs-treated IVDs showed higher normalized NZ stiffness compared to damaged IVDs.

Under creep conditions, damaged IVDs exhibited increased normalized creep displacements, indicating a decrease in normalized creep elastic stiffness.

The results show that reducing GAG content in damaged IVDs increases mechanical flexibility, but eEV therapy prevents the loss of glycosaminoglycans and subsequent changes in mechanical function.

FOXF1 eEVs induced structural and functional changes in IVD by increasing proteoglycan and GAG levels.

The results of the study showed that eEVs loaded with developmental transcription factors can treat painful joint diseases such as DBP by delivering these transcription factors into degenerative and painful joints by IVD.

This strategy may help reduce the structural and functional abnormalities caused by the disease and regulate pain responses in a gender-specific manner.

The researchers also recommended using developmental transcription factors such as FOXF1 to shift degenerative NP cells into a pro-anabolic state in vivo. Further studies are needed to determine its therapeutic efficacy.