title: Nucleus pulposus cellular longevity by telomerase gene therapy.
abstract: METHODS
  Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties.
  
  
  OBJECTIVE
  To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner.
  
  
  BACKGROUND
  Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process.
  
  
  METHODS
  Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed.
  
  
  RESULTS
  Transfection was achieved successfully with 340% +/- 7% (mean +/- SD) relative telomerase activity in hTERT-transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions.
  
  
  CONCLUSIONS
  Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency, or G1-cell cycle checkpoint control was evident.
authors.affiliation: Orthopaedic Research Institute, University of New South Wales, St. George Hospital Campus, NSW, Australia.
authors.forename: Sylvia A
authors.initials: SA
authors.lastname: Chung
authors.forename: Ai Qun
authors.initials: AQ
authors.lastname: Wei
authors.forename: David E
authors.initials: DE
authors.lastname: Connor
authors.forename: Graham C
authors.initials: GC
authors.lastname: Webb
authors.forename: Timothy
authors.initials: T
authors.lastname: Molloy
authors.forename: Marina
authors.initials: M
authors.lastname: Pajic
authors.forename: Ashish D
authors.initials: AD
authors.lastname: Diwan
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics; D002869:Chromosome Aberrations; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D008055:Lipids; D012333:RNA, Messenger / Q000378:metabolism; D012756:Sheep; D013122:Spinal Diseases / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D003094:Collagen; C509186:TERT protein, human; D019098:Telomerase