Validation Of A Genetically-modified Autologous Adipocyte Cell Therapy For Peptide Delivery
Mengfan Wu, MD, PhD1, Ziyu Chen, MD, PhD1, John McNamara, BS2, Shailesh Agarwal, MD1.
1Harvard Medical School, Boston, MA, USA, 2Rutgers University, Newark, NJ, USA.
PURPOSE: Adipose autografts are often used as structural fillers. Despite being highly cellular tissues, adipose autografts have not been leveraged as agents for the production or delivery of therapeutic agents. Adipocytes present a promising a cell therapy due to their abundance in most individuals. We hypothesized that adipocytes can be purified from adipose tissues and modified to express and secrete therapeutic peptides of interest.
METHODS: Adipocytes were isolated from human extra-abdominal fat (n=3) using a novel collagenase-free method using a MACS tissue dissociator (include one additional detail here) (Fig A). Plasmids encoding peptide inhibitors of bone morphogenetic protein (BMP) or transforming growth factor beta (TGFβ): CMV-BMPR1AFc or CMV-TGFβR2Fc respectively were constructed. Electroporation (500V/5 msec/4 pulses) was used to deliver the desired plasmids (n = 3). Adipocytes were cultured for 28 days in vitro in DMEM/10% FBS/1% pen-strep. Normalized (Act2b) transgene expression (BMPR1AFc or TGFβR2Fc) was quantified using mRNA isolated from adipocytes; IgG-Fc ELISA was performed using media from adipocytes to quantify secreted levels. Co-culture experiments were performed to test bioactivity of secreted BMPR1AFc and TGFβR2Fc based on down-regulation of BMP- (Col10) or TGFβ-mediated (Col1a1, Fibronectin) genes. Finally, engraftment of electroporated adipocytes was tested with subcutaneous delivery of red fluorescent adipocytes derived from red fluorescent C57BL/B6 mice into wild type C57BL/6 mice.
RESULTS: Human adipocytes were isolated by mechanical, collagenase-free method and cultured up to 28 days. qPCR verified significantly increased expression of BMPR1AFc and TGFβR2Fc in adipocytes electroporated with the desired plasmid (2.8 x 10^7 v. 1.0, p < 0.05 and 2.2 x 10^7 v. 1.0, p < 0.05 respectively). IgG Fc ELISA verified secretion of BMPR1A-Fc and TGFβRII-Fc in adipocytes electroporated with the desired plasmid relative to adipocytes electroporated without plasmid (Day 7: BMPR1A-Fc: 62 ng/mL v. 44 ng/mL, p<0.05; TGFβR2-Fc: 66 ng/mL v. 44 ng/mL, p<0.05 and Day 14: BMPR1A-Fc: 40 ng/mL v. 24 ng/mL, p<0.05; TGFβR2-Fc: 47 ng/mL v. 24 ng/mL, p<0.05) (Fig A). Co-culture assays confirmed bioactivity of secreted agents (BMPR1A-Fc: Col10 0.06 v. 1.0, p<0.05 and TGFβRII-Fc: Col1a1 0.50 v. 1.0, p < 0.01, Fibronectin 0.45 v. 1.0 p < 0.05). Finally, mouse adipocytes successfully engrafted in the subcutaneous location following electroporation, based on presence of adipose tissue at the site of injection with characteristic red fluorescence under microscopy (Fig B/C).
CONCLUSION: Our findings demonstrate that adipocytes can be harvested from adipose tissue in a 20-minute protocol yielding a highly-purified population. These adipocytes are capable of peptide expression and secretion with functional activity of the tested agents. Using the IgG-Fc ELISA, we noted background secretion of detectible peptide which is likely due to non-specific antibody binding. That these electroporated adipocytes engraft and survive points to their potential as an endogenous source of therapeutic agents. Future work will include development of a strategy for genetic permanence using transposase/transposon delivery and for regulated expression using inducible or niche-specific promoters.
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