PDGFB-Based Stem Cell Gene Therapy Increases Bone Strength in the Mouse

PDGFB-based stem cell gene therapy increases bone PNAS PLUS strength in the mouse Wanqiu Chena, David J. Baylinka, Justin Brier-Jonesa, Amanda Neisesa, Jason B. Kiroyana, Charles H. Rundlea,b, Kin-Hing William Laua,b, and Xiao-Bing Zhanga,1 aDepartment of Medicine, Loma Linda University, Loma Linda, CA 92354; and bMusculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, Loma Linda, CA 92357 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 5, 2015 (received for review January 27, 2015) Substantial advances have been made in the past two decades in (HSC) cell therapy, which could be given intravenously and would the management of osteoporosis. However, none of the current result in rejuvenation of the skeleton (9). We have shown en- medications can eliminate the risk of fracture and rejuvenate the graftment of donor HSCs that were genetically engineered to skeleton. To this end, we recently reported that transplantation overexpress FGF2 at sites where bone is lost in osteoporosis (i.e., + of hematopoietic stem/progenitor cells (HSCs) or Sca1 cells engi- the HSC niches), which in turn resulted in substantial augmenta- neered to overexpress FGF2 results in a significant increase in la- tion of bone matrix formation at these sites (9). Despite these mellar bone matrix formation at the endosteum; but this increase advances, we encountered several issues that severely compro- was attended by the development of secondary hyperparathyroid- mised the efficacy of our therapy. Instead of being stronger, the ism and severe osteomalacia. Here we switch the therapeutic gene resulting bones were actually weaker and sometimes fractured PDGFB to , another potent mitogen for mesenchymal stem cells during tissue processing. This was associated with severe hypo- (MSCs) but potentially safer than FGF2. We found that modest calcemia, secondary hyperparathyroidism, and osteomalacia de- PDGFB overexpression of using a relatively weak phosphoglycer- veloped in response to the therapy. ate kinase (PGK) promoter completely avoided osteomalacia and In the present study, we sought to resolve these adverse side secondary hyperparathyroidism, and simultaneously increased effects. Two changes in our therapy were made compared with trabecular bone formation and trabecular connectivity, and de- our previous work. First, we switched the therapeutic gene from creased cortical porosity. These effects led to a 45% increase FGF2 to PDGFB, because PDGF-BB is normally secreted after in the bone strength. Transplantation of PGK-PDGFB–transduced + platelet activation and circulates at concentrations that are some Sca1 cells increased MSC proliferation, raising the possibility that 100-times higher than FGF2 (and thus could be safer). We use PDGF-BB enhances expansion of MSC in the vicinity of the hema- the term PDGFB to refer the gene that encodes the human topoietic niche where the osteogenic milieu propels the differen- tiation of MSCs toward an osteogenic destination. Our therapy platelet-derived growth factor (PDGF) B chain, and the term should have potential clinical applications for patients undergoing PDGF-BB to refer the homodimeric protein. PDGF-BB is a HSC transplantation, who are at high risk for osteoporosis and major growth factor found in bone matrix (10) and has also been bone fractures after total body irradiation preconditioning. It shown to increase bone formation after intravenous adminis- could eventually have wider application once the therapy can be tration (11). In addition, there have been considerable successful – applied without the preconditioning. applications of PDGF-BB based therapies on various types of maladies, including tendon, periodontal ligament, and bone PDGFB | hematopoietic stem cells | bone formation | gene therapy fracture repairs (12, 13). The safety of PDGF-BB has been demonstrated in several clinical trials (14, 15), and it has been steoporosis is a major public health problem in the United approved by the Food and Drug Administration for treatment of OStates and in the world. Currently, there are almost 10 million patients with oral and maxillofacial bony defects (15). Second, osteoporosis-related fractures annually worldwide (1). Over the we used various promoters of different strengths to express past two decades, the treatment of osteoporosis has advanced PDGFB to identify the optimal PDGF-BB dosage. In the present dramatically, primarily because of the successful development of several effective antiresorptive therapies (2), which can reduce the Significance fracture rate by as much as 50% (3). An anabolic therapy, namely parathyroid hormone (PTH), was subsequently developed and Osteoporosis is a morbid disease afflicting millions of people approved by the Food and Drug Administration. This therapy worldwide. To unlock the unique regenerative powers of the increases bone formation as opposed to antiresorptive drugs that skeleton that have not yet been exploited, we used stem cell gene MEDICAL SCIENCES reduce bone resorption, but the efficacy of this anabolic therapy therapy to dramatically increase bone formation at sites where in terms of fracture reduction has been the same as that of bone is lost during osteoporosis. Our therapy tremendously in- antiresorptive drugs (4). Multiple newer and more potent anabolic creased de novo trabecular bone formation and trabecular con- agents are currently being evaluated in clinical trials (5–7), but nections, resulting in a large increase in bone strength. Our none of these entities appear to have the potential to rejuvenate therapy has clinical potential, may serve as a prototype for future the osteoporotic skeleton back to one with normal bone density skeletal stem cell gene therapies, and is a model for mechanistic and strength. studies of de novo trabecular bone formation. All of the aforementioned medications fall into the realm of pharmaceutical or biologic therapies. However, we have now Author contributions: D.J.B., K.-H.W.L., and X.-B.Z. designed research; W.C., J.B.-J., A.N., J.B.K., entered the era of the third pillar of medicine: cell therapy (8). and C.H.R. performed research; J.B.-J. contributed new reagents/analytic tools; W.C., C.H.R., and X.-B.Z. analyzed data; and W.C., D.J.B., K.-H.W.L., and X.-B.Z. wrote the paper. “Cells uniquely sense their surroundings, make decisions and Conflict of interest statement: A provisional patent application has been filed on the basis exhibit varied and regulable behaviors,” such as targeting (8). In of some of the findings. D.J.B., K.-H.W.L., X.-B.Z., and W.C. are the coinventors. this regard, we have initiated the development of an anabolic cell This article is a PNAS Direct Submission. therapy for the skeleton (9). Our past work has focused on 1To whom correspondence should be addressed. Email: [email protected]. establishing proof-of-principle for this approach in the mouse This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. model, using genetically engineered hematopoietic stem/progenitor 1073/pnas.1501759112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501759112 PNAS | Published online July 6, 2015 | E3893–E3900 Downloaded by guest on September 28, 2021 study, we showed that when a relatively weak physiologic pro- Optimal PDGFB Expression Driven by a Weaker Promoter Promotes moter (i.e., phosphoglycerate kinase or PGK promoter) was Endosteal/Trabecular Bone Formation Without Osteomalacia or used, the therapy yielded marked increases in endosteal/trabecular Secondary Hyperparathyroidism. Because mice with low-level se- bone formation without significant elevation in the circulating rum PDGF-BB showed higher BMD, we decided to reduce level of PDGF-BB. It also avoided adverse effects, such as oste- PDGFB expression by using two weaker promoters: that is, the omalacia, and led to a 45% increase in bone strength (maximal PGK promoter (∼500 bp in length) and the truncated PGK load to failure). In this respect, no traditional monotherapy for promoter, P200 (∼200 bp in length) (Fig. S1). The P200 trun- osteoporosis has shown long-term beneficial effect on the bone cated promoter is less potent compared with the original PGK promoter. To assess relative potency of these different pro- strength of the treated bone (16). Moreover, our therapy uniquely + + produced a 20-fold increase in trabecular connectivity and sub- moters in Sca1 cells, we used GFP as the reporter gene. Sca1 stantial reduction in cortical porosity, both of which are relevant to cells were transduced with the Lenti SFFV-GFP, PGK-GFP, or the bone mechanical performance. P200-GFP, each at the same low multiplicity of infection (MOI) of 0.1. A low MOI was used because it would allow a better Results comparison of the transduction efficiency among strong and + weak promoters. As shown in Fig. 2A, the transduction efficiency Transplantation with Lenti-SFFV-PDGFB–Transduced Sca1 Cells Leads + to Massive Endosteal/Trabecular Bone Formation but also Induces for the three test vectors in Sca1 cells was very similar (i.e., ∼4% in each transduced cell population). As expected, the level Osteomalacia. Our cell-based therapy consists of isolation of ∼ HSCs from a donor mouse, genetically engineering these cells of GFP expression driven by the PGK promoter was 85% lower to overexpress PDGFB, and transplantation of these modified than that driven by the SFFV promoter (15% vs. 100%), and the GFP expression level controlled by the P200 promoter was HSCs into a syngeneic recipient mouse after irradiation (Fig. 1A) ∼50% lower than PGK promoter (9% vs. 15%) (Fig. 2B). (9). HSCs are used as a vehicle because these cells specifically To investigate the dosage effects of PDGF-BB on bone for- target to the HSC niche at the endosteal bone surface and the + mation, we transduced Sca1 cells with Lenti SFFV-PDGFB, HSC niches at the marrow vascular surfaces: the two major sites PGK-PDGFB, P200-PDGFB, or GFP control (each at an MOI of bone loss in humans with osteoporosis.

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