The effect of cell-mediated delivery of combination VEGF165, GDNF, and NCAM1 genes on molecular and cellular reactions in the spinal cord of pigs with contusion trauma



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Abstract

Currently, the treatments for spinal cord injury are limited. Gene therapy is one of the most promising approaches aimed at overcoming negative post-traumatic consequences in the spinal cord. Numerous studies performed in rodents indicate a positive effect of the delivery of therapeutic genes to the spinal cord to stimulate neuroregeneration. However, to bring the developed protocols of gene therapy to the stage of clinical trials, it is necessary to verify the results obtained in experiments on large laboratory animals. Objective: Immunofluorescence analysis of the response of markers of cell stress and apoptosis, synaptic proteins and neuroglia in the spinal cord of female vietnamese pot-bellied pigs after intrathecal delivery of genes encoding vascular endothelial growth factor (VEGF165), glial-derived neurotrophic factor and neuronal cell adhesion molecule (NCAM1), using human umbilical cord blood mononuclear cells (UCBMC). In experimental pigs (n = 2), 4 hours after modeling a dosed contusion injury of the spinal cord at the Th8-Th9 level, 2х106 genetically modified UCBMCs overexpressing recombinant VEGF, GDNF, and NCAM molecules in 200 |jl of saline were intrathecally injected. Control animals (n = 2) were injected with 200 jl of saline into the cerebrospinal fluid. Intact pigs (n = 2) were used to obtain baseline values for immunofluorescence analysis of post-traumatic molecular and cellular responses. After 60 days, immunofluorescence analysis in the rostral and caudal parts of the spinal cord relative to the epicenter of injury revealed positive changes in experimental pigs against the background of cell-mediated delivery of the VeGf165, GDNF, and NCAM1 genes. In the anterior horns of the rostral and caudal spinal cord of animals from the therapeutic group, a higher level of fluorescence of the synaptic protein synaptophysin, a lower number of astrocytes and microglial cells were found, which may indicate functional recovery of neurons and suppression of the development of astrogliosis. In the rostral section, in the area of the corticospinal tract, gene therapy maintained the number of oligodendrocytes, which ensure myelination of regenerating axons. The results obtained suggest that genetically modified UCBMCs, overexpressing recombinant molecules VEGF and GDNF (as therapeutic molecules) and NCAM (as a molecule providing survival and targeted targeting of cell carriers), contribute to post-traumatic regeneration of the spinal cord.

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About the authors

M. A Davleeva

Kazan state medical university

F. V Bashirov

Kazan state medical university

A. A Izmailov

Kazan state medical university

F. O Fadeev

Kazan state medical university

M. E Sokolov

Kazan state medical university

V. A Markosyan

Kazan state medical university

R. R Garifulin

Kazan state medical university

M. S Kuznetsov

Kazan state medical university

I. A Pakhalina

Kazan state medical university

I. S Minyazeva

Kazan state medical university

Yu. A Chelyshev

Kazan state medical university

R. R Islamov

Kazan state medical university

References

  1. O'Shea T.M., Burda J.E., Sofroniew M.V. Cell biology of spinal cord injury and repair. The Journal of clinical investigation. Am. Soc. Clin. Inv. 2017; 127(9): 3259-70.
  2. Petrosyan H.A., Alessi V., Hunanyan A.S. et al. Spinal electromagnetic stimulation combined with transgene delivery of neurotrophin NT-3 and exercise: novel combination therapy for spinal contusion injury. J. Neurophysiol. 2015; 114(5): 2923-40.
  3. Ahuja C.S., Fehlings M. Concise Review: Bridging the Gap: Novel Neuroregenerative and Neuroprotective Strategies in Spinal Cord Injury. STEM CELLS Transl. Med. 2016; 5(7): 914-24.
  4. Badner A., Siddiqui A.M., Fehlings M.G. Spinal cord injuries: how could cell therapy help? Expert Opinion on Biol. Ther. 2017; 17(5): 529-41.
  5. Thuret S., Moon L.D.F., Gage F.H. Therapeutic interventions after spinal cord injury. Nature reviews. Neuroscience 2006; 7(8): 628-43.
  6. Boekhoff T.M., Flieshardt C., Ensinger E.M. et al. Quantitative magnetic resonance imaging characteristics: evaluation of prognostic value in the dog as a translational model for spinal cord injury. J. Spinal disorders & Techniques 2012; 25(3): E81-7.
  7. Zurita M., Aguayo C., Bonilla C. et al. The pig model of chronic paraplegia: A challenge for experimental studies in spinal cord injury. Progress in Neurobiol. Prog. Neurobiol. 2012; 97(3): 288-303.
  8. Jones C.F., Lee J.H.T., Kwon B.K. et al. Development of a large-animal model to measure dynamic cerebrospinal fluid pressure during spinal cord injury: Laboratory investigation. J. Neurosurg. 2012; 16(6): 624-35.
  9. Lee J.H.T., Jones C.F., Okon E.B. et al. A Novel Porcine Model of Traumatic Thoracic Spinal Cord Injury. J. Neurotrauma 2013; 30(3): 142-59.
  10. James N.D., Shea J., Muir E.M. et al. Chondroitinase gene therapy improves upper limb function following cervical contusion injury. Exp. Neurol. 2015; 271: 131-5.
  11. Walthers C.M., Seidlits S.K. Gene delivery strategies to promote spinal cord repair. Biomarker Insights 2015; 2015 Suppl 1: 11-29.
  12. Hodgetts S.I., Harvey A.R. Neurotrophic Factors Used to Treat Spinal Cord Injury. Vitamins and Hormones 2017; 104: 405-57.
  13. Chen J., Bernreuther C., Dihne M. Cell adhesion molecule l1-trans-fected embryonic stem cells with enhanced survival support regrowth of corticospinal tract axons in mice after spinal cord injury. J. Neurotrauma 2005; 22(8): 896-906.
  14. Krakora D., Mulcrone P., Meyer M. et al. Synergistic effects of GDNF and VEGF on lifespan and disease progression in a familial ALS rat model. Mol. Ther. 2013; 21(8): 1602-10.
  15. Izmailov A.A., Povysheva T.V., Bashirov F.V. et al. Spinal Cord Molecular and Cellular Changes Induced by Adenoviral Vector- and Cell-Mediated Triple Gene Therapy after Severe Contusion. Frontiers in Pharmacol. 2017; 8: 813.
  16. Islamov R.R., Izmailov A.A., Sokolov M.E. et al. Evaluation of direct and cell-mediated triple-gene therapy in spinal cord injury in rats. Brain Res. Bulletin 2017; 132: 44-52.
  17. Islamov R.R., Rizvanov A.A., Fedotova V.Y. et al. Tandem Delivery of Multiple Therapeutic Genes Using Umbilical Cord Blood Cells Improves Symptomatic Outcomes in ALS. Mol. Neurobiology 2017; 54(6): 4756-63.
  18. Измайлов А.А., Соколов М.Е., Баширов Ф.В. и др. Сравнительный анализ эффективности прямой и клеточно-опосредованной генной терапии крыс с контузионной травмой спинного мозга. Гены и Клетки 2018; XII(4): 53-9.
  19. Фадеев Ф.О., Баширов Ф.В., Измайлов А.А. и др. Нейроглия при контузионной травме спинного мозга крысы на фоне клеточно-опосредованной доставки комбинации генов VEGF165, GDNF и NCAM1 в сочетании с эпидуральной электрической стимуляцией. Гены и Клетки 2020; XV(2): 58-65.
  20. Yang W.Z., Zhang Y., Wu F. et al. Safety evaluation of allogeneic umbilical cord blood mononuclear cell therapy for degenerative conditions. J. Transl. Med. 2010; 8(1): 1-6.
  21. Neuhoff S., Moers J., Rieks M. et al. Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro. Exp. Hematol. 2007; 35(7): 1119-31.
  22. Fan C.G., Zhang Q.J., Tang F.W. Human umbilical cord blood cells express neurotrophic factors. Neuroscience Letters 2005; 380(3): 322-5.
  23. Islamov R.R., Sokolov M.E., Bashirov F.V. et al. A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs. Neuroscience Letters 2017; 644: 67-75.
  24. Shcherbinin D.N., Esmagambetov I.B., Noskov A.N. et al. Protective immune response against Bacillus anthracis induced by intranasal introduction of a recombinant adenovirus expressing the protective antigen fused to the Fc-fragment of IgG2a. Acta Naturae 2014; 6(1): 76-84.
  25. Islamov R.R., Rizvanov A.A., Mukhamedyarov M.A. et al. Symptomatic improvement, increased life-span and sustained cell homing in amyotrophic lateral sclerosis after transplantation of human umbilical cord blood cells genetically modified with adeno-viral vectors expressing a neuro-protective factor and a neur. Current Gene Therapy 2015; 15(3): 266-76.
  26. Riley J., Federici T., Park J. et al. Cervical spinal cord therapeutics delivery: Preclinical safety validation of a stabilized microinjection platform. Neurosurg. 2009; 65(4): 754-61.
  27. Liddelow S.A., Guttenplan K.A., Clarke L.E. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 2017; 541(7638): 481-7.
  28. Zhu H., Poon W., Liu Y. et al. Phase III Clinical Trial Assessing Safety and Efficacy of Umbilical Cord Blood Mononuclear Cell Transplant Therapy of Chronic Complete Spinal Cord Injury. Cell Transplantation 2016; 25(11): 1925-43.
  29. Liu J., Han D., Wang Z. et al. Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells. Cytotherapy 2013; 15(2): 185-91.
  30. Yao L.Q., He C., Zhao Y. et al. Human umbilical cord blood stem cell transplantation for the treatment of chronic spinal cord injury: Electrophysiological changes and long-term efficacy. Neural Reg. Res. 2013; 8(5): 397-403.

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