Autonomous Bmp9 Ex Vivo Gene Therapy Reverses Lymphedema By Promoting Functional Lymphatic Regeneration

Background Lymphedema is a chronic disorder characterized by impaired lymphatic drainage and interstitial fluid accumulation, affecting an estimated 200-250 million people worldwide. Therapeutic approaches that restore lymphatic function are urgently needed. Bone morphogenetic protein 9 (BMP9), a member of the TGF-β family that signals through the ALK1 receptor, has been implicated in lymphovascular maturation and valve formation during embryogenesis. However, its role in post-natal lymphangiogenesis remains unknown. We hypothesized that BMP9 can rescue sites of lymphedema through functional lymphatic regeneration. Furthermore, we sought to develop a strategy for endogenous, autonomous Bmp9 production at the affected site. Methods A hindlimb lymphedema model was generated in mice by surgical ablation of lymphatic vessels and regional lymph nodes. Mice were intraperitoneally treated with vehicle, recombinant BMP9 (50 ng/mouse), or ALK1-Fc (50 ng/mouse), a soluble decoy receptor that blocks BMP9/10 signaling. Lymphedema severity was assessed by limb circumference and thickness of skin layer, lymphatic regeneration by LYVE1+Podoplanin+ immunofluorescence, and adipose tissue remodeling by Oil Red O staining. To identify candidate promoters for driving Bmp9 expression, we examined previously-published RNA transcriptome sequencing (GSE302563) on subcutaneous tissue from ISL stage III lymphedema patients (n = 4) and normal controls (n = 3). A transposon plasmid containing the HIF1A promoter was engineered to enable Bmp9 expression in response to elevated VEGF-C levels. HEK293 cells transfected with this VEGF-C-inducible Bmp9 plasmid were cultured in VEGF-C-supplemented media for 7 days, and qPCR confirmed enhanced Bmp9 expression. 3 days after lymphedema induction, adipocytes transduced with this plasmid were subcutaneously injected into the affected site. 14 days post-injection, tissue thickness and lymphatic drainage were evaluated by intradermal injection of FITC-dextran (2,000 kDa, 10 μL) into the footpad, followed by axillary lymph node imaging and FITC/Area quantification. Results BMP9 treatment significantly reduced lymphedema swelling (two-way ANOVA, p 0.0001) and skin thickness compared to control and ALK1-Fc groups ( A-C ). BMP9 increased lymphatic vessel density (p = 0.0027), whereas ALK1-Fc abolished these effects (p 0.0001) ( D ). BMP9 also reduced adipose accumulation, while ALK1-Fc exacerbated it ( E ). RNA sequencing revealed upregulation of HIF1A (1.4-fold, p = 0.012) and FOXC2 (21-fold, p = 0.010) in lymphedema tissue ( F ). The HIF1A promoter was thus selected for plasmid construction. In HEK293 cells, VEGF-C (25-50 ng/mL) induced a 6- to 28-fold increase in Bmp9 expression (p 0.05) ( G,H ). Mice treated with transfected Bmp9 -expressing adipocytes showed markedly reduced skin thickness and a 7.8-fold higher normalized FITC intensity (p = 0.0220), indicating enhanced lymphatic drainage ( I,J ). Conclusions BMP9 promotes lymphatic regeneration and mitigates pathological remodeling, including skin thickening and adipose accumulation, in hindlimb lymphedema. Furthermore, the validation of a VEGF-C-inducible Bmp9 expression system supports the feasibility of localized, durable gene delivery. Autonomous ex vivo Bmp9 gene therapy using adipocytes effectively enhances lymphatic drainage and restores lymphedematous tissues, highlighting its potential as a regenerative therapeutic strategy for lymphedema. *Source: https://ps-rc.org/meeting/Program/2026/04.cgi*