Bringing gene therapy to regenerative medicine

Abstract

Gene therapy has its intellectual origins in the context of treating Mendelian disorders,1Friedmann T. Roblin R. Gene therapy for human genetic disease?.Science. 1972; 175: 949-955https://doi.org/10.1126/science.175.4025.949Google Scholar with the fruits of its decades-long development reflected in the increasing number of gene therapeutics gaining regulatory approval. However, single-gene defects are rare. The potential scope of gene therapy was greatly broadened with the realization that it had potential application in common, complex conditions that lack a monogenetic basis. In this context, gene transfer serves as a drug delivery system for encoded therapeutic proteins and non-coding species of RNA. This application was first appreciated in the case of arthritis, and several arthritis gene therapies are now in clinical trials.2Evans C.H. Ghivizzani S.C. Robbins P.D. Osteoarthritis gene therapy in 2022.Curr. Opin. Rheumatol. 2023; 35: 37-43https://doi.org/10.1097/BOR.0000000000000918Google Scholar This provided the impetus for expansion into the field of regenerative medicine.3Evans C.H. Huard J. Gene therapy approaches to regenerating the musculoskeletal system.Nat. Rev. Rheumatol. 2015; 11: 234-242https://doi.org/10.1038/nrrheum.2015.28Google Scholar Regenerative medicine is an attractive application of gene therapy because most of the morphogens and growth factors of interest for this purpose are difficult to deliver to sites of tissue damage and have very short biological half-lives. Gene transfer offers the prospect of providing these proteins by local synthesis in a sustained fashion at concentrations sufficient to drive a robust regenerative process. Problems in bone healing provided the earliest application of this concept,4Baltzer A.W. Lattermann C. Whalen J.D. Wooley P. Weiss K. Grimm M. Ghivizzani S.C. Robbins P.D. Evans C.H. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.Gene Ther. 2000; 7: 734-739https://doi.org/10.1038/sj.gt.3301166Google Scholar,5Lieberman J.R. Le L.Q. Wu L. Finerman G.A. Berk A. Witte O.N. Stevenson S. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.J. Orthop. Res. 1998; 16: 330-339https://doi.org/10.1002/jor.1100160309Google Scholar which remains the area where most progress has been made. Although bone is one of the few organs in the human body that can regenerate scarlessly, in about 5%–10% of cases, fractures fail to heal, leading to non-unions. These are recalcitrant clinical problems. The 1965 publication by Marshall Urist6Urist M.R. Bone: formation by autoinduction.Science. 1965; 150: 893-899https://doi.org/10.1126/science.150.3698.893Google Scholar of a bone inductive extract with the ability to induce new endochondral bone formation revolutionized our understanding of bone biology. In the era preceding modern molecular technology, it took decades to isolate, purify, and eventually clone these molecules, which became known as bone morphogenetic proteins or BMPs.7Sampath T.K. Reddi A.H. Discovery of bone morphogenetic proteins - a historical perspective.Bone. 2020; 140: 115548https://doi.org/10.1016/j.bone.2020.115548Google Scholar Once cloned, BMPs became available at scale, thereby enabling the first clinical use of BMPs in non-unions, which dates to the late 1980s. Recombinant, human BMP-2 (rhBMP-2) is available for clinical use in the United States and Europe as part of a product known as Infuse™ Bone Graft, whose approved indications are high-energy open tibial fractures, spinal fusions, and certain oromaxillofacial applications. Additionally, it has wide off-label use8Ong K.L. Villarraga M.L. Lau E. Carreon L.Y. Kurtz S.M. Glassman S.D. Off-label use of bone morphogenetic proteins in the United States using administrative data.Spine. 2010; 35: 1794-1800https://doi.org/10.1097/BRS.0b013e3181ecf6e4Google Scholar in treating osseous non-unions and a related condition where a large segment of bone is missing, usually as a result of trauma. Beyond a certain critical size, segmental defects in bone never heal spontaneously and can lead to amputation. Despite much promise, the clinical efficacy of rhBMP-2 has been disappointing. In attempts to improve outcomes, very high amounts of the protein are used, but these provoke numerous adverse side effects, some of them severe.9James A.W. LaChaud G. Shen J. Asatrian G. Nguyen V. Zhang X. Ting K. Soo C. A review of the clinical side effects of bone morphogenetic protein-2.Tissue Eng. B Rev. 2016; 22: 284-297https://doi.org/10.1089/ten.TEB.2015.0357Google Scholar Moreover, recent evidence suggests that bone formed in response to large amounts of rhBMP-2 is of inferior quality.10Panos J.A. Coenen M.J. Nagelli C.V. McGlinch E.B. Atasoy-Zeybek A. Lopez De Padilla C. De la Vega R.E. Evans C.H. Segmental defect healing in the presence or absence of recombinant human BMP2: novel insights from a rat model.J. Orthop. Res. 2023; https://doi.org/10.1002/jor.25530Google Scholar Much of the blame for the mediocre clinical response to rhBMP-2 has focused on inadequate delivery from the collagen sponge used as a scaffold, and there has been considerable research into developing improved scaffolds that deliver higher amounts of rhBMP-2 locally for longer periods of time. Gene transfer was proposed as a superior technology for achieving this end. Genetic delivery of BMP-2 indeed proved superior to protein delivery of rhBMP-2 in pre-clinical models of bone healing, but in an unexpected way—efficient healing via gene therapy occurred when only small amounts of BMP-2 were expressed in vivo for a short period of time.11De la Vega R.E. Atasoy-Zeybek A. Panos J.A. VAN Griensven M. Evans C.H. Balmayor E.R. Gene therapy for bone healing: lessons learned and new approaches.Transl. Res. 2021; 236: 1-16https://doi.org/10.1016/j.trsl.2021.04.009Google Scholar As described in this issue of Molecular Therapy – Methods and Clinical Development, Chris Evans and his team investigated why this was so.12Atasoy-Zeybek A. Coenen M.J. Hawse G.P. Logeart-Avramoglou D. Evans C.H. De La Vega R.E. Efficient autocrine and paracrine signaling explain the osteogenic superiority of transgenic BMP-2 over rhBMP-2.Mol. Ther. Methods Clin. Dev. 2023; 29: 350-363https://doi.org/10.1016/j.omtm.2023.03.017Google Scholar This in vitro study focused on BMP-2 signaling via SMAD phosphorylation using reporter murine mesenchymal cells where firefly luciferase expression is driven by elements of the Id1 promotor, an early response gene activated by BMP-2.13Logeart-Avramoglou D. Bourguignon M. Oudina K. Ten Dijke P. Petite H. An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct.Anal. Biochem. 2006; 349: 78-86https://doi.org/10.1016/j.ab.2005.10.030Google Scholar Responses to BMP-2 synthesized endogenously as a result of transduction with adenovirus (Ad.BMP2) or lentivirus (LV.BMP2) were compared with those elicited by exogenously delivered rhBMP-2. The study confirmed the superiority of transgenic BMP-2 cDNA over rhBMP-2, showing that the former was at least 100-fold more effective in signaling. Interestingly, when analyzing the amount of BMP-2 produced by transduced cells, the authors found that over 30% of the BMP-2 remained cell associated; this was more pronounced (i.e., over 50%) when AdBMP2 was used. This was associated with partial resistance to noggin, a physiological inhibitor that binds to BMP-2. The secreted amounts of endogenously synthesized BMP-2 were very low, and media conditioned by the transduced cells expressing BMP-2 were unable to induce luciferase expression in naive reporter cells. Co-culture experiments demonstrated that cells expressing BMP-2 were able to induce luciferase expression in adjacent naive cells. When BMP-2 cells were co-cultured with naive cells but separated by a permeable insert, there was no activation of the naive cells, suggesting that close proximity and possibly cell-to-cell contact was necessary for this to occur. To confirm that these data were not a result of using an established cell line, osteogenesis of primary cultures of human mesenchymal stromal cells was demonstrated. In summary, the authors explain the superior activity of transgenic BMP-2 by its ability to elicit powerful autocrine and paracrine effects in a protected pericellular environment. Although the study focused on SMAD signaling via the BMP type 1A receptor, the authors left open the possibility of additional pathways, including intracrine signaling based on the observation that a substantial portion of transgenic BMP-2 remained cell associated. These data provide mechanistic support for the use of gene therapy-based approaches to regenerating bone and provide, for the first time, a plausible explanation for the observation that gene transfer is superior to a bolus of protein in healing bone. The efficiency of tiny amounts of transiently expressed, endogenously synthesized BMP-2 in healing bone suggests novel opportunities beyond traditional gene therapy. Recently, messenger RNA (mRNA) has shown remarkable success in pre-clinical models of bone healing.14De La Vega R.E. van Griensven M. Zhang W. Coenen M.J. Nagelli C.V. Panos J.A. Peniche Silva C.J. Geiger J. Plank C. Evans C.H. Balmayor E.R. Efficient healing of large osseous segmental defects using optimized chemically modified messenger RNA encoding BMP-2.Sci. Adv. 2022; 8: eabl6242https://doi.org/10.1126/sciadv.abl6242Google Scholar A stable and non-immunogenic formulation of mRNA encoding BMP-2 efficiently healed critical-size bone defects in rats, with the regenerate rapidly achieving superior biomechanics and undergoing advanced tissue remodeling. Based on these encouraging findings, mRNA therapeutics promise to revolutionize the application of gene therapy to regenerative medicine by combining safety, efficacy, and affordability.15Balmayor E.R. Synthetic mRNA - emerging new class of drug for tissue regeneration.Curr. Opin. Biotechnol. 2022; 74: 8-14https://doi.org/10.1016/j.copbio.2021.10.015Google Scholar The author has no competing interests to declare.