Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates upon rAAV-Mediated Gene Transfer and Overexpression of the Insulin-Like Growth Factor I

Abstract

Direct therapeutic gene transfer in marrow concentrates is an attractive strategy to conveniently enhance the chondrogenic differentiation processes as a means to improve the healing response of damaged articular cartilage upon reimplantation in sites of injury. In the present study, we evaluated the ability of the clinically adapted recombinant adeno-associated virus (rAAV) vectors to mediate overexpression of the insulin-like growth factor I (IGF-I) in human bone marrow aspirates that may modulate the proliferative, anabolic activities, and chondrogenic differentiation potential in such samples in vitro. The results demonstrate that successful, significant rAAV-mediated IGF-I gene transfer and expression were achieved in transduced aspirates (up to 105.9 -35.1 pg rhIGF-I/mg total proteins) over time (21 days) at very high levels (*80% of cells expressing the candidate IGF-I transgene), leading to increased levels of proliferation, matrix synthesis, and chondrogenic differentiation over time compared with the control (lacZ) condition. Treatment with the candidate IGF-I vector also stimulated the hypertrophic and osteogenic differentiation processes in the aspirates, suggesting that the regulation of IGF-I expression through rAAV will be a prerequisite for future translation of the approach in vivo. However, these findings show the possible benefits of this vector class to directly modify marrow concentrates as a convenient tool for strategies that aim at improving the repair of articular cartilage lesions.

Peripheral blood aspirates overexpressing IGF‐I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes

“…Peripheral blood (~3 ml) was collected in the presence of hirudin from donors of the Department of Orthopaedic Surgery, Saarland University Medical Center, Homburg/Saar, Germany ( n = 4; age 42 ± 27), with informed consent. All procedures were in accordance with the Helsinki Declaration.…”

“…All constructs applied in the study were based on pSSV9, an AAV‐2 genomic clone . rAAV‐ lacZ carries the E. coli β‐galactosidase (β‐gal) gene and rAAV‐hIGF‐I a human insulin‐like growth factor I (hIGF‐I) cDNA (536 bp) both under the control of the cytomegalovirus immediate–early (CMV‐IE) promoter. The 293 adenovirus‐transformed embryonic kidney cell line was used to package conventional (not self‐complementary) recombinant vectors (rAAV) with helper functions provided by Adenovirus 5 and the pAd8 helper plasmid .…”

“…TGF‐β, basic fibroblast growth factor, i.e . FGF‐2, IGF‐I) and transcription factors (SOX9) for chondrogenic purposes, thus far there is little evidence on the possibility of targeting human peripheral blood aspirates with this vector class. We were actually the first group to provide evidence that overexpression of TGF‐β via rAAV significantly enhanced the chondrogenic differentiation processes in such samples .…”

“…Yet, as the treatment also stimulated hypertrophy and osteogenic differentiation events, new avenues of research to identify other candidate genes are needed for a safe translation of the strategy in future in vivo settings. In the light, the mitogenic and pro‐anabolic IGF‐I factor may be a good alternative as it displays chondroreparative activities when applied to human BM‐MSCs as isolated or concentrated populations as well as in articular chondrocytes . The goal of this study was thus to investigate the effects of overexpressing IGF‐I via rAAV upon the chondrogenic differentiation processes in human peripheral blood aspirates.…”

Genetic Modification of Human Peripheral Blood Aspirates Using Recombinant Adeno-Associated Viral Vectors for Articular Cartilage Repair with a Focus on Chondrogenic Transforming Growth Factor-β Gene Delivery

“…In this regard, administration of recombinant adeno-associated viral (rAAV) vectors might be best suited because these constructs are clinically adapted, promoting high and persistent levels of transgene expression in various human musculoskeletal cell populations [12][13][14][15][16][17][18] compared with other less efficient or more immunogenic/toxic vectors (nonviral, adenoviral, retroviral/ lentiviral vectors) [19][20][21][22][23][24][25][26]. Although chondrogenic gene transfer has been successfully performed via rAAV in aspirates from the bone marrow of patients by using transforming growth factorb (TGF-b) [27], insulin-like growth factor-I (IGF-I) [28], and the cartilage-specific SOX9 transcription factor [29], no evidence thus far demonstrates the possibility of modifying human peripheral blood aspirates with this vector class. The goal of the present study was therefore to evaluate the ability of rAAV to directly transduce human peripheral blood aspirates, with a focus on delivering a TGF-b gene as a means to stimulate the chondrogenic differentiation processes in such samples compared with control treatments.…”