Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic

Abstract

The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.

PEGylated Liposomes Associate With Wnt3A Protein and Expand Putative Stem Cells in Human Bone Marrow Populations

“…Moreover, PEGylation had no effect on the activity of DOPC liposomes (p = 0.65). In agreement with previous studies [28], we found that 24 h of incubation of Wnt3A with either PEGylated or unPEGylated liposomes was sufficient to partition the majority of the Wnt3A protein into the liposome fraction. >80% of protein was found in the pellet fraction of ultracentrifuged liposome suspension at this time point, measured by a protein quantification assay and western blotting (Figure 3C & D).…”

“…In contrast, in our experiments, we found that activity of Wnt3A in both unPEGylated and PEGylated DOPC liposomes was preserved, and that PEGylation of DMPC liposomes reduced activity by only approximately 50%. This was dependent on the addition of Wnt post extrusion, and was time dependent, with increases in activity upon incubation for up to 24 h, as has been shown for DMPC liposomes previously [28]. There are several possible reasons why certain lipids may inhibit Wnt activity -these include low incorporation of protein in liposomes during fabrication; denaturation during the incorporation process; incorrect presentation of protein to recipient cells; or insufficient interaction/ binding of liposomes to target cell populations.…”

“…As Wnt proteins carry fatty acid modifications which render them unstable in aqueous solution [26,27], liposome incorporation (for one member of the Wnt family, Wnt3A) has been shown to be an effective method of preserving both in vitro and in vivo stability and activity [28][29][30][31][32]. However, several challenges and unanswered questions remain.…”

Wnt3a Reestablishes Osteogenic Capacity to Bone Grafts from Aged Animals

“…We solved this in vivo delivery problem by packaging the hydrophobic Wnt3a in lipid particles. This formulation of the human Wnt3a protein, liposomal Wnt3a (L-Wnt3a), is stable in vivo 49 and promotes robust bone regeneration in a modified fracture model 31 . Although exogenously applied Wnt3a has great potential as a therapeutic protein, safety remains a primary concern.…”

Regenerative Medicine: Charting a New Course in Wound Healing

“…An important example is the Wnt/beta-catenin signaling pathway that regulates the expression of genes critical for embryonic development and adult stem cell biology and mutations, which are associated with cancer. 57,58 Investigators are attempting to selectively influence this pathway using many of the approaches described here: Wnt proteins delivered using novel strategies, 59 gene-based strategies such as microRNAs and small inhibitory RNA transgenes, and small-molecule inhibitors. 60 Figure 2 illustrates the many points along the Wnt pathway that can be targeted with regenerative technologies.…”