In a recent study published in Biomaterials, researchers developed a novel non-viral gene therapy for discogenic back pain (DBP) by delivering a developmental-type transcription factor, Forkhead Box F1 (FOXF1), using engineered extracellular vesicles (eEVs) to the degenerative intervertebral disc (IVD) in vivo.
Study: Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain. Image Credit: Natali _ Mis/Shutterstok.com
Background
Chronic low back pain (LBP) is a growing global issue due to aging populations 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 techniques, including growth factor administration, cell-based therapeutics, and viral genetic delivery treatments, can reduce degeneration in animal and human models.
However, concerns like fleeting effects, poor long-term effectiveness, and unnecessary immunogenicity and tumorigenicity may prevent direct translation.
About the study
In the present study, researchers established a non-viral gene therapy for intervertebral disc (IVD) degeneration using FOXF1-eEVs.
The researchers transfected primary mouse embryonic fibroblasts (PMEFs) with a plasmid containing FOXF1 or pCMV6 as a control and characterized eEV samples using nanoparticle tracking analysis (NTA).
They assessed the effective loading of the molecular payload within eEVs using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and conventional PCR.
Western blot analysis determined FOXF1 and specific EV proteins in eEV formulations. The team used plasmids amplifying upstream and downstream polylinker regions to ascertain the presence of FOXF1 plasmid deoxyribonucleic acid (DNA) in donor cells and generated eEVs.
They examined the full-length messenger ribonucleic acid produced from plasmid DNA in eEVs and donor cells.
The researchers created extracellular vesicles with transcription factors to restore tissue function and modify pain responses in an animal model of DBP.
They defined the EVs to transport and distribute FOXF1 to damaged intervertebral discs in the lumbar discal puncture discogenic back pain murine model to determine FOXF1 eEV inhibition on intervertebral discal degeneration.
The team coupled biomechanical testing of mice’s IVD joints with imaging, extracellular matrix (ECM) alterations, and pain behaviors evaluated at 12 weeks to validate changes in structure and function and pain caused by the therapeutic intervention.
Pre-operation and post-treatment pain assessments included micro-computed tomography (micro-CT), magnetic resonance imaging (MRI), mechanical tests, Alcian Blue (AB) and Picrosirius Red (PSR) staining, the Dimethyl Methylene Assay, and immunohistochemistry (IHC).
The study featured a surgical technique in which the researchers subcutaneously injected Buprenorphine ER into mice to control post-operative pain.
The team performed behavioral assessments pre-operatively and biweekly from four to twelve weeks after surgery, using various modalities such as open field, cold plate, tail suspension, and hanging wire.
The open field test evaluated spontaneous mouse activity; cold plate tests measured thermal hyperalgesia; tail suspension assays measured axial pain; and hanging wire assays measured strength.
Twelve weeks after surgery, the team dissected the lumbar spine of the animals, using femoral nerve and artery tracing to identify the intervertebral discs between L4 and L5, L5 and L6, and L6 and S1 IVDs. They used L5/L6 IVD to assess histology and determine glycosaminoglycan (GAG) content.
Results
FOXF1 eEVs considerably decreased pain behaviors while restoring IVD structure and function, including enhanced disc height, tissue hydration, proteoglycan content, and mechanical characteristics.
The study focused on FOXF1-loaded eEV release from primary fibroblasts transfected with the developmental transcription factor FOXF1. Quantitative RT PCR indicated a considerable increase in FOXF1 mRNA transcripts and full-length transcribed FOXF1 mRNA levels compared to pCMV6-transfected cells.
FOXF1 eEV therapy might reduce pain behavior in a mouse lumbar disc puncture model for up to 12 weeks. Female mice demonstrated a higher grip time for FOXF1-treated groups than the damaged group, which lasted at least 12 weeks after treatment.
FOXF1 eEV therapy improved IVD tissue hydration and height in damaged and degenerate animals in vivo while preserving hydration levels and T2-weighted IVD disc intensity.
However, the team observed reduced disc height in wounded and pCMV6 eEV-treated animals. FOXF1 eEV-treated mice exhibited no reduction in disc height 12 weeks after treatment. There were no sex impacts on functional results.
FOXF1 eEVs restored mechanical function to damaged and degenerated IVDs in vivo. Under axial stress, IVDs treated with FOXF1 eEVs exhibited higher normalized NZ stiffness than wounded IVDs.
Under creep circumstances, wounded IVDs demonstrated increased normalized creep displacements, indicating reduced normalized creep elastic stiffness.
The findings indicate that decreasing GAG content in damaged IVDs enhanced mechanical compliance, but eEV therapy prevented glycosaminoglycan loss and consequent mechanical functional alterations.
FOXF1 eEVs induced structural and functional alterations in the IVD via raising proteoglycan and GAG levels.
Conclusion
The study findings revealed that EVs loaded with developmental transcription factors could treat painful joint illnesses such as DBP by delivering these transcription factors to degenerated and painful IVD joints.
This strategy can help attenuate structural and functional abnormalities caused by the disease while also regulating pain behaviors based on sex.
The researchers also advocated employing developmental transcription factors such as FOXF1 to convert degenerate NP cells into a pro-anabolic state in vivo. Further research is needed to determine its therapeutic efficacy.
