Abstract
Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 μg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 μg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.
INTRODUCTION
Large osseous segmental defects do not heal well and remain a major clinical problem that can lead to amputation. Regenerative medicine offers new possibilities for restoring lost bone and producing a regenerate that is indistinguishable from its native counterpart. The identification and cloning of morphogens that guide the behavior of osteoprogenitor cells provide opportunities for biologically based tissue regeneration.Recombinant, human bone morphogenetic protein-2 (rhBMP-2) and rhBMP-7 have been approved for use in the United States and Europe, but the clinical efficacy of these molecules has been disappointing and adverse side effects have been associated with their use (1). The latter are exacerbated, and the financial cost increased, by the very high amounts of recombinant protein that need to be applied to overcome the inadequacies of the collagen sponge used as a scaffold for protein delivery. Several approaches to improving protein delivery, including the use of smart scaffolds (2), have been investigated. However, none of these have advanced to clinical trials. Furthermore, rhBMP-7 has been withdrawn from the market and is no longer available for clinical use. Alongside, restrictions have been imposed in the clinical use of rhBMP-2, and this product is not available in many countries.Local gene delivery provides a promising, alternative approach to delivering osteogenic proteins at therapeutic levels within an osseous lesion for an extended period of time (3). Successful intralesional delivery and expression of complementary DNA (cDNA) encoding an osteogenic product such as a BMP will result in the sustained in situ production of authentic BMP by the endogenous biological machinery of the cell. Substantial preclinical progress has been made (3, 4), and the results confirm that low amounts of BMP-2 synthesized endogenously within the osseous lesion for a short period of time by genetically modified cells efficiently heal bone (3). Despite these encouraging results, traditional gene therapy has yet to generate a clinical product. The reasons for this are several including the disadvantages of current genetic delivery strategies, cost, safety concerns, translational barriers, and the regulatory complexity of advancing gene therapies for non-Mendelian, non–life-threatening conditions.Taking the above into consideration, messenger RNA (mRNA), the intermediary between a gene and its encoded protein, may offer the best of both worlds and confer numerous advantages. After delivery into a cell, mRNA will almost immediately begin to be translated into its cognate protein, unlike the case with traditional gene delivery of DNA, which needs first to translocate to the nucleus of the cell before its expression can begin. The protein encoded by mRNA is expressed from the introduced message for a finite period of time, after which the mRNA is degraded by innate physiological processes of the cell, leaving no residue. There is therefore no risk of insertional mutagenesis or other genetic damage, unlike the case with DNA therapeutics. Moreover, synthetic mRNAs encoding specific therapeutic proteins are relatively easy and straightforward to produce and can be readily delivered into the cell with inexpensive, nonviral vectors. Together, these advantages endow mRNA therapeutics with several important advantages over their DNA equivalents. They are expected to be safer and easier to translate to a large patient population, as demonstrated by the mRNA vaccines in use against coronavirus disease 2019 (COVID-19).The application of mRNA to regenerative medicine has been hampered by its instability, cytotoxicity, and inflammatory properties (5). However, these limitations can be overcome by transcript engineering. Modifications that have been investigated to affect the stability and biocompatibility of mRNA include changes in the 5′ and 3′ untranslated regions (UTRs), open reading frame alterations, poly(A) tail length, and the use of modified nucleosides (6–10). The latter markedly reduce immunogenicity (11, 12). Recent advances in mRNA technology, led by these structural modifications and by the use of lipid vectors for delivery, have kindled interest in the use of mRNA in regenerative medicine as a safe and effective means of tissue restoration (13).Here, we have evaluated the ability of an optimized chemically modified mRNA (cmRNA) encoding BMP-2 (12) to heal a critical-sized segmental defect in the rat femur. The data demonstrate the complete healing of the damaged tissue with almost no escape of the cmRNA from the lesion into the circulation or major organs. De novo formed bone showed mechanical properties comparable to those of the native bone while lacking the massive callus formed by rhBMP-2 in this model. Unlike the latter, BMP-2 cmRNA induced bone healing via the native endochondral route. The work summarized here demonstrates the efficacy and safety of an affordable transcript therapy for expedited bone healing.
RESULTS
Local, intraosseous expression of cmRNA without escape from the site of application
Figure S1 summarizes the study design. We used a standardized rat, mid-diaphyseal, femoral, 5-mm defect model stabilized by a bridging plate (fig. S1B). If left untreated, this defect does not heal. Intralesional implantation of 50 μg of cmRNA encoding firefly luciferase (FLuc) on a collagen sponge resulted in orthotopically localized expression of FLuc at a declining rate for at least 3 days (Fig. 1, A to D). No luciferase activity was detected when a truncated, noncoding (NC) cmRNA was administered (Fig. 1, C and D). No expression of luciferase was detected at other sites in the body by in vivo imaging (Fig. 1A)….
