Bone or Cartilage Disease

Coding Dimension ID: 
279
Coding Dimension path name: 
Bone or Cartilage Disease

Cartilage Regeneration by the Chondrogenic Small Molecule PRO1 during Osteoarthritis

Funding Type: 
Early Translational II
Grant Number: 
TR2-01829
ICOC Funds Committed: 
$6 792 660
Disease Focus: 
Arthritis
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Other
oldStatus: 
Active
Public Abstract: 
The ability to direct the differentiation of resident mesenchymal stem cells (MSCs) towards the cartilage lineage offers considerable promise for the regeneration of articular cartilage after traumatic joint injury or age-related osteoarthritis (OA). MSCs can be stimulated in vitro to form new functional cartilage. In the OA-affected joint, the repair is insufficient, leaving a damaged matrix, suggesting that key factors are missing to properly direct the regenerative process. Molecules that activate the chondrogenic potential of cartilage stem cells may potentially prevent further cartilage destruction and stimulate repair of cartilage lesions. Currently there are no disease-modifying therapeutics available for the 40 million Americans suffering from OA. Therapeutic options are limited to oral and intra-articularly injected pain medications and joint replacement surgery. The primary objective of this project is to develop a non-invasive, therapeutic for the regeneration of cartilage in OA. This new therapy will target the resident MSCs in the joint, stimulate production of new cartilage matrix, promote repair and thus limit additional joint damage and improve joint pain and function. To provide a proof-of-concept for our strategy, a cell-based screen of a diverse small molecule library led to compounds capable of enhancing the formation of articular cartilage (chondrogenesis) from MSCs in vitro. In secondary assays, molecules were assessed for protection of the existing cartilage against induced tissue damage. Through these approaches, the lead low molecular weight small molecule PRO1 was identified which promotes cartilage differentiation and protects cartilage from damage. PRO1 reproducibly demonstrated in vivo efficacy in two animal models of OA (surgical and enzyme-induced). OA-associated pain was reduced and the architecture of the cartilage was restored. PRO1 therefore appears to activate the regenerative potential of the resident cartilage stem cells.
Statement of Benefit to California: 
Osteoarthritis (OA) is the most prevalent musculoskeletal disease and globally the 4th leading cause of Years Lost to Disease (YLD). OA affects over 40 million Americans and the magnitude of the problem is predicted to increase even further with the obesity epidemic and aging of the baby boomer generation. It is estimated that 80% of the population will have radiographic evidence of OA by age 65 years. The annual economic impact of arthritis in the U.S. is estimated at over $100 billion, representing more than 2% of the gross domestic product. OA accounts for 25% of visits to primary care physicians. In 2004 OA patients received 650,000 knee and hip replacements at a cost of $26 billion. Without change in treatment options 1.8 million joint replacements will be performed in 2015. OA is a painful, degenerative type of arthritis; physical activity can become difficult or impossible. Some patients with osteoarthritis are forced to stop working because their condition becomes so limiting. OA can interfere with a patient's ability to even perform routine daily activities, resulting in a decrease in quality of life. The goals of osteoarthritis treatment are to relieve pain and other symptoms, preserve or improve joint function, and reduce physical disability. Current therapeutic options are limited to pain medications and joint replacement for patients with advanced disease. No disease-modifying OA drugs are approved for clinical use. OA is thus a major unmet medical need with a huge clinical and socioeconomic impact and a complete absence of effective therapies. Clearly the development of a new therapeutic that is both symptom and disease modifying would have a significant impact on the well-being of Californians and reduce the negative economic impact on the state resulting from this highly prevalent disease.
Progress Report: 
  • We have carried out a structure-activity relationship study to identify highly potent analogues of kartogenin with chondrogenic and chondroprotective activities. Over 150 analogues were synthesized with structurally diverse elements and assessed for chondrogenic activity (ability to induce mesenchymal stem cells to differentiate into cartilage producing chondrocytes) on human and rodent mesenchymal stem cells. A number of highly potent lead compounds were identified which will next be assessed in chondroprotective assays, cell-based selectively and toxicity assays, pharmacokinetic assays and in vivo rodent efficacy models. At the same time a number of assays were developed and used to assess the chondrocyte protective effects, joint retention, and proliferative activity on human chondrocytes of the parent compound, kartogenin. Kartogenin was found to: (1) have long term human and rodent chondrogenic activity; (2) possess chondroprotective activity in bovine chondrocytes (i.e., protects against degradative activities in the joint); (3) minimally induce chondrocytes proliferation (an undesired activity that could lead to fibrotic and immune responses); (4) have good joint retention (compound retained in the intra-articular space at the site of action); and (5) is subject to rapid systemic clearance (a desirable property to minimize systemic adverse effects).
  • We also identified the mechanism by which the compound functions. In contrast to other drugs in development for osteoarthritis, kartogenin does not target extracellular enzymes involved in joint cartilage degradation. Rather it appears to act directly on an endogenous stem cell population and induce chondrocyte formation. The molecule binds selectively to an intracellular protein filamin A, a protein involved in regulating the cell’s cytoskeletal network (structural elements inside the cell). Rather than modulating the interaction of filamin A with other structural proteins, kartogenin blocks its interaction with the protein CBFβ (core binding factor β subunit, a subunit of a transcription complex with the runt-related transcription factor (RUNX) family). The result is an increase in CBFβ levels in the nucleus where it binds and activates transcription of RUNX dependent genes. In particular CBFβ activates RUNX1 dependent transcription of genes that play key roles in chondrogenesis. Thus this molecule acts by a novel mechanism directly and selectively on gene transcription to induce the selective differentiation of mesenchymal stem cells to chondrocytes. Importantly molecules that act by this method should complement the activity of drugs in clinical trials aimed at blocking degradative enzymes.
  • We have made excellent progress toward the identification of a preclinical candidate for the treatment of osteoarthritis. A large structure-activity relationship study was carried out with the chemical synthesis of over 250 analogues of the original lead compound. We have identified molecules with improved activity in cell culture and in relevant preclinical in vivo models. Based on these efforts we are synthesizing a final series of molecules which we will profile with respect to in vitro and in vivo chondrogenesis activity, pharmacokinetics and safety. We expect to choose the final preclinical candidate from this series in the third year of the grant.
  • Osteoarthritis (OA) is the most prevalent musculoskeletal disease affecting about 27 million people in the United States, and is the leading cause of chronic disability in the United States. Current therapeutic options are limited to pain or symptom-modifying drugs and joint replacement surgery; no disease-modifying drugs are approved for clinical use. OA is characterized by progressive degeneration of the articular cartilage, resulting from abnormal activation, differentiation and death of cartilage cells. A unique and unexplored therapeutic opportunity exists to induce somatic stem cells to regenerate the damaged tissue and reverse the chronic destructive process. Because limited joints are affected in most OA patients, intra-articular (IA) drug injection is an attractive treatment approach that allows high local drug concentration with limited systemic exposure. Targeting resident stem cells pharmacologically also avoids the risks and costs associated with cell-based approaches.
  • Cartilage contains resident mesenchymal stem cells (MSCs) that can be differentiated in vitro to form chondrocytes. This observation suggests that intra-articular injection of a small molecule that promotes chondrogenesis in vivo will preserve and regenerate cartilage in OA-affected joints. Using an image-based screen, we identified a drug-like small molecule, kartogenin (KGN), that promotes efficient and selective chondrocyte differentiation from MSCs in vitro. Intra-articular injection of KGN also shows beneficial effects in surgery-induced acute and enzyme-induced chronic cartilage injury models in rodents, as well as positive effects in incapacitance pain models. This project is aimed at the development of new lead compounds with improved biological activity, the demonstration of efficacy of the lead compounds in rodent and dog OA models and the elucidation of the cellular mechanisms underlying the cartilage regeneration activities of KGN and its analogs.
  • Through medicinal chemistry efforts, we have designed and synthesized over 400 analogs of KGN. Using cell culture based assays, we assessed the chondrocyte differentiation activity of these analogs and identified 17 compounds exhibiting improved potency compared to KGN (EC50 < 100 nM). These compounds showed no obvious cytotoxicity at high concentrations (100 μM) when incubated with a variety of cells present in the joints including MSCs, chondrocytes, osteoblasts and synoviocytes. Up to date, we have assessed the efficacy of 7 compounds using a rat OA model (medial meniscal tear). Two of the tested compounds showed significantly improved cartilage repair at the end of the study. At the same time, no adverse effects, such as body weight loss, pain or impaired motor functions, were observed in any compound treated animals. We are currently studying the effects of another 10 analogs using the same OA model, which is expected to conclude within two to three months. Next, we will assess the efficacy of active compounds in a canine OA model (partial meniscectomy using beagles). Furthermore, full rodent pharmacokinetics and non-GLP toxicology studies will be performed for the lead compounds.
  • To study the underlying mechanisms of KGN induced chondrogenesis, we designed and synthesized an affinity probe with biological activities comparable to that of KGN. Through affinity-based methods, we identified protein filamin A (FLNA) as the target of KGN. In MSCs, KGN binds to FLNA and disrupts its interaction with core binding factor β (CBFβ), which leads to the nuclear translocalization of CBFβ, subsequent activation of the RUNX1-CBFβ transcription program and, as a result, chondrocyte differentiation. This mechanism has been confirmed using cell biological methods including RNAi mediated gene silencing and cDNA overexpression of relevant genes such as FLNA, CBFβ and RUNX1. These studies have been published in the journal Science.
  • We have demonstrated efficacy in preclinical in vivo models of a potential drug candidate for the treatment of osteoarthritis. The small molecule functions by selectively differentiating meschenchymal stem cells to chondrocytes to repair damaged cartilage .

Gene Targeting to Endogenous Stem Cells for Segmental Bone Fracture Healing

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06713
ICOC Funds Committed: 
$5 185 487
Disease Focus: 
Arthritis
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Segmental bone fractures are a complex medical condition. These injuries cause great suffering to patients, long-term hospitalization, repeated surgeries, loss of working days, and considerable costs to the health system. It is well known that bone grafts taken from the patient (autografts) are considered the gold-standard therapy for these bone defects. Yet these grafts are not always available, and their harvest often leads to prolonged pain. Allografts, are "dead" bone grafts, which are readily available from tissue banks, but have very low potential to induce bone repair. We have previously shown that stem cells from human bone marrow, engineered with a bone-forming gene, can lead to complete repair of segmental fractures. However, such an approach requires several steps, which could complicate and prolong the pathway to clinical use. An alternative approach would be to gene-modify stem cells that already reside in the fracture site. We were the first to show, in a rodent model, that a segmental bone defect can be completely repaired by recruitment stem cells to the defect site followed by direct gene delivery. In the proposed project we aim to further promote this approach to clinical studies. The project will include the development of a direct gene delivery technology, based on ultrasound. We will test the efficiency of the method in repairing large bone defects and its safety. If successful, we will be able to proceed to FDA approval towards first-in-human trials.
Statement of Benefit to California: 
Segmental bone defects are a complex medical problem that often requires bone grafting. Autografts are considered the gold standard for these defects, but their usage is limited by availability and donor-site morbidity and supply. Allografts are more available but often fail to integrate with the host bone. Thus there is an unmet need in the field of orthopedic medicine for novel therapies for segmental bone fractures. We propose to develop a novel approach for the treatment of such fractures without the need for a bone graft. Specifically, we will utilize ultrasound to deliver a bone-forming gene to stem cells that will be recruited to the defect site. As we have already shown, the gene would trigger the cells to regenerate the bone that had been lost due to trauma or cancer. If successful, this project could lead to the development of a simple treatment for massive bone loss. Such a treatment will benefit the citizens of California by reducing loss of workdays, duration of hospital stays, and operative costs, and by improving quality of life for Californians with complex segmental bone fractures.
Progress Report: 
  • Segmental bone fractures constitute a complex medical condition with no effective treatment. These injuries cause great suffering to patients, long-term hospitalization, repeated surgeries, loss of working days, and considerable costs to the health system. It is well known that autologous bone grafts (autografts) that are harvested from the patient, are considered the gold-standard therapy for these bone defects. Yet these grafts are not always available, and their harvest often leads to prolonged postoperative pain and comorbidity at the donor site. Bone allografts obtained from tissue banks are readily available, but lead to poor graft-host integration resulting in numerous failures. We have previously shown that mesenchymal stem cells (MSCs) engineered with a specific bone-forming gene can be used to achieve complete regeneration of segmental fractures in long bones. However, such an approach requires several steps—cell isolation, expansion, and engineering—which could complicate and prolong the regulatory pathway to clinical use. An alternative approach would be to gene-modify endogenous stem cells that reside within in the body. We were the first to show, in a rodent model, that a segmental bone defect can be completely repaired by recruitment of endogenous stem cells to the fracture site followed by direct gene delivery. In this research project we aimed to further promote this therapeutic approach to clinical studies. During the first year of the project we investigated the use of an ultrasound system to deliver genes to feature sites. Our results showed that we were able to deliver the genes to 40-50% of the cells residing in the fracture site. Moreover, 70-90% of the cells that received the genes were identified as stem cells, which are the target of the therapeutic approach. Our next goal would be to deliver bone-forming genes to stem cells in the fracture site in order to induce complete defect repair.

Tissue engineered cartilage from autologous, dermis-isolated, adult, stem (DIAS) cells

Funding Type: 
Early Translational III
Grant Number: 
TR3-05709
ICOC Funds Committed: 
$1 735 703
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
This study addresses the cartilage defects resulting from injuries or from wear-and-tear that can eventually degenerate to osteoarthritis. This is a significant problem that impacts millions and costs in excess of $65B per annum in the US alone. Addressing this indication successfully holds potential for halting the progression of cartilage damage before it destroys the entire joint. We have shown that articular cartilage can be engineered with properties on par with native tissues using chondrocytes. Also, skin derived stem cells can be used to engineer new cartilage with significant mechanical integrity. Combining these findings, the new cellular therapy that this proposal seeks to develop is an autologous skin cell-derived combination product for articular cartilage repair. Three aims are proposed to advance this autologous, adult stem cell-based method: First, protocols shown to be efficacious in cartilage tissue engineering will be applied to skin-derived stem cells and show safety in the mouse model. Then, using a preclinical model, the desired biological response, toxicology, and durability will be verified. Finally, short-term safety and efficacy of cartilage repair will be examined in a different preclinical model. Successful completion of this DCF project will allow the start of preclinical studies in the sheep that demonstrate long-term safety and efficacy, as specified by the FDA.
Statement of Benefit to California: 
Arthritis is the leading cause of disability in the US, affecting over 46 million Americans. Of these, over 5 million Californians are affected by this debilitating disease, with roughly 3 million that are women and over 2 million that are men. Additionally, Californian youth is also included in the estimated 30 million children who participate in organized sports activities, whose yearly costs for injuries have been projected to be $1.8 billion. For young patients with knee injuries, 75% exhibit superficial (grade I–II) and 25% exhibit deep (grade III–IV) cartilage lesions. Young patients not only need to retain mobility for many years in life but also new, tissue-sparing techniques. This proposal seeks to develop an autologous, adult stem cell-based therapy that addresses grade II-IV cartilage lesions. The source of these cells will be the skin, using minimally invasive procedures. The development of such a therapy would expand the clinical options available to Californians. The assembled team of academics, orthopaedic surgeons, and veterinary surgeons are based in the [REDACTED]. The refinement of this research will not only benefit [REDACTED] in terms of increasing competitiveness for NIH funding, but it will also allow for Californian companies to license the technology and therefore benefit economically.
Progress Report: 
  • Cartilage degeneration resulting from injuries or wear-and-tear leads to osteoarthritis, which impacts millions and costs in excess of $65B per annum. No long-term solutions exist for cartilage degeneration, but cellular therapies hold promise toward replacing degenerated cartilage with healthy tissue. This Development Candidate Feasibility Award is a first step toward the overall goal of developing a cell-based cartilage repair therapy using stem cells derived from the skin. The therapy would consist of using a skin biopsy to harvest dermis-isolated, adult stem cells (DIAS cells), which will undergo processing to yield neocartilage. This neocartilage will then be implanted into the patient’s joint to restore or improve mobility.
  • Work during this progress report period has been divided into project preparation and scientific progress. Project preparation includes setting up facilities and approvals for work with human DIAS cells, identifying sources and acquiring human skin for DIAS cell isolation, and hiring and training personnel. Scientific progress includes a publication on co-cultures using stem cells, work on culturing larger numbers of cells using low oxygen tension, comparing stem cells from human skin of different anatomical locations, and gaining an understanding of the niches where skin stem cells may reside.
  • The project now has a consistent source of human dermis tissue from which stem cells can be isolated. This includes skin containing hair follicles and also skin without follicles. Spherical culture of human skin-derived stem cells has been performed. It was found that directing stem cells into cells that make cartilaginous matrix can be more efficacious if done under low oxygen tension. Since much of the prior work on directing stem cells from the skin to form neocartilage has been done using animal-derived stem cells, in the next project period neocartilage will be formed using human stem cells instead. Technologies developed using animal models can thus be translated toward human use.

Harnessing native fat-residing stem cells for bone regeneration

Funding Type: 
Early Translational II
Grant Number: 
TR2-01821
ICOC Funds Committed: 
$5 391 560
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Other
oldStatus: 
Active
Public Abstract: 
Like most tissues of the body, bone possesses a natural regenerative system aimed at restoring cells and tissues lost to natural cell aging, disease or injury. These natural regenerative systems are complex combinations of cell growth factors and support structures that guide and control the development of specialized bone stem cells. However, the regeneration process may still fail, for multiple reasons. For instance, the degree of skeletal injury may be so great that it overwhelms the natural regenerative capacity. Alternatively, the natural regenerative capacity may be defective; this is exemplified by osteoporosis, a frequent condition affecting post-menopausal females and elderly males and females. Osteoporotic individuals have severe declines in stem cell numbers (10-fold decrease from age 30 to 80) and stem cell function (tendency of stem cells to turn into fat rather than bone cells with age), leading to bone loss and “fragility fractures” that typically would not occur in persons with normal stem cell number and function. Thus, there is a tremendous need for therapies to increase the number and function of endogenous adult stem cells with the potential to build new bone. One option is to introduce so called mesenchymal stem cells (MSC) from the patient to bone repair sites. However, significant hurdles to autologous MSC use include the need for 2-3 week culture times to isolate MSC before application. Moreover culturing introduces infectious and immunogenic risks from prolonged exposure to animal products and cancerous risks from cellular gene changes in culture. In addition, once isolated, MSC require appropriate growth factor stimulation to form bone. Finally, MSC isolated from patient tissues such as fat or bone marrow are heterogenous and of undetermined composition—making growth factor dosing and conformance with FDA regulations for defining target product identity, purity, and potency more difficult. To circumvent these problems, we have identified and purified the cells at the origin of human MSC. We have termed these perivascular stem cells (PSC) because they are natively localized around all arteries and veins, forming the key cellular component of the natural regenerative system. In a significant breakthrough, we are able to isolate these cells within hours from adipose tissues in sufficient numbers for therapy without the need for culture. This realizes the possibility of harvesting and implanting stem cells during the same operative period. In another breakthrough, we have identified a potent growth factor NELL-1 that potently amplifies the ability of PSC to form bone and vascular structures. This has led to the development of our target PSC+NELL-1 product, which effectively stimulates and augments the body’s natural bone regenerative system by providing all the components (stem cells, growth factor, and allograft bone support structure) necessary to “jump start” as well as maintain the function of bone stem cells.
Statement of Benefit to California: 
This FDA oriented proposal focuses on crucial preparatory work required before IND-enabling preclinical studies on our Developmental Candidate for bone formation and regeneration. Our Developmental Candidate provides a complete package of stem cells, bone growth factors, and scaffold to build an optimized microenvironment to “jump start” bone formation in normal and impaired bone healing conditions. We have generated very promising preclinical data on our Developmental Candidate’s superior bone formation and regeneration efficacy. In addition to its significant impact on health care, this highly multi-disciplinary project by our team has many near-term and long-term benefits to the State of California. 1. Besides direct health costs, musculoskeletal injuries and diseases are the leading cause of work-related and physical disability in the United States. Hard working Californians are responsible for California’s annual gross domestic product of $1.8 trillion, which rank our state among the top eight largest economies in the world. By promoting the repair of both normal and healing-impaired bone in a safe and effective manner, our mature technology will reduce the loss of work productivity at the front end, reduce work disability costs, and reduce the loss of state income tax. 2. Local osteogenic stem cells decline with age (from 1/10,000 in newborns to 1/250,000 by age 30 and 1/2,000,000 by age 80), leading to osteoporosis, poor bone quality, and fragility fractures. In 1998, the health care burden for osteoporosis exceeded $2.4 billion in California alone. A whopping 64% of the $2.4 billion was caused by hip fracture. If our Development Candidate is successful in healing existing fractures in impaired bone, it may also translate to therapies to prevent fractures in impaired bone. This will significantly reduce the long-term health care burden for California’s public health insurance program. 3. This project directly adds jobs at [REDACTED] and at the California-based companies that are involved in this project. 4. This project will produce intellectual property that is owned by t [REDACTED]. Our team has a track record of attracting out of state private investment to invest in California and of procuring supplies and equipment from strategic California-based companies. 5. This mature project is precisely the type of cutting-edge, multi-disciplinary stem cell project that Californians imagined when they approved proposition 71 in 2004. The establishment of CIRM has transformed the research infrastructure at [REDACTED], increased our ability to recruit world class stem cell scientists, and attracted the attention of superb scientists from other disciplines to this new field. Working together, our team has compiled an impressive list of accomplishments and we are confident in our abilities to take this project to IND submission in a timely fashion. Funding of this project will fulfill the promise of proposition 71.
Progress Report: 
  • Background: unsuccessful bone repair
  • Most organs can regenerate cells lost to ageing, exposure to adverse conditions, ephemeral lifespan, disease or injury. Regenerative systems are complex combinations of growth factors and anchorage molecules which support, guide and control the maturation of specialized stem cells. The regenerative process may still fail, for multiple reasons. Bone fractures will sometimes not heal spontaneously because parts of the broken bone do not join anymore, or because bone regeneration itself is impaired, like in osteoporosis, a frequent condition that affects post-menopause females and elderly people of both genders.
  • A novel class of bone-healing stem cells
  • Since specialized bone-forming cells have not been identified, which stem cells could be used for the cell therapy of bone fractures? We have identified and purified novel stem cells, localized around blood vessels and therefore named perivascular stem cells (PSCs). We have also demonstrated that Nell-1, a potent osteogenic growth factor we have developed, efficiently turns PSCs into bone cells. In 2011, we have been granted support by the California Institute for Regenerative Medicine to develop a product to heal critically fractured bones in osteoporotic – or not – patients or to perform spine fusion in order to correct skeletal defects. PSCs will be purified from the patient’s own fat tissue, a well-documented, rich source of these cells, and embedded in a biocompatible scaffold in the presence of the bone forming growth factor. The resulting compound, inserted at the site of the fracture, will provide bone-forming stem cells 1-precisely characterized in terms of origin and identity; 2- derived from the patient, hence not rejected; 3- not cultured, therefore similar to their natural counterparts.
  • Progress after one year of research supported by CIRM
  • We are now close to the end of the first year of our CIRM supported studies. In this initial period we have, first, validated the stem cells of use with regard to stringent biologic criteria: how many of these cells can be purified from fat tissue? How viable and pure are these cells after the sorting process? Do stem cell numbers and quality depend on the age, sex and corpulence of the donor? Following analysis of about 80 distinct fat harvests (lipoaspirates) we have determined that robust bone forming PSC can be reliably purified, in sufficient numbers, from male and female donors in all age and weight ranges. We have also shown that PSC are at least as good, in terms of bone forming potential, than conventional mesenchymal stem cells (the candidate stem cells, so far, to be used for cell therapies of bone defects amd injuries), while being significantly superior regarding purity and safety.
  • Transplantation experiments in mice and rats have revealed that the combination of PSC and the Nell-1 growth factor ensures the most efficient bone regeneration, but that PSC do not give rise to tumors, an important verification in any protocol involving transplantation. These experiments have also demonstrated that PSC stimulate the formation of new blood vessels, an essential requisite for efficient wound healing and tissue repair.
  • Finally, we have embarked on the development of a larger animal model of bone regeneration, that will be used in the second and third years of the projects.
  • In summary, we have reached all the milestones and met the deadlines planned in the original project. The next year will see the further validation of the protocol and the beginning of the development of a stem cell/scaffold/growth factor combination product in the perspective of trials in human patients.
  • Our research group has identified in the human body a novel class of stem cells endowed with strong osteogenic potential, which means capacity to produce bone. These stem cells are attached to the outer aspect of blood vessels, and therefore present in all organs. Our ultimate goal is to use these stem cells in human beings, to perform for instance spine fusion, a procedure used to treat spine deformities (scoliosis) or faulty vertebrae, or to heal complicated bone fractures, especially in patients suffering from osteoporosis, a condition where spontaneous bone repair is compromised. We shall harvest stem cells from the patient’s own abdominal adipose (fat) tissue, a rich source of these cells which is also easy and safe to collect. Stem cells will be purified on an automated machine, named fluorescence activated cell sorter, and immobilized on an engineered biocompatible scaffold in the presence of a novel osteogenic growth factor, that is a sort of hormone which stimulates stem cell development into bone. We have won an Early Translational grant (2011-2014) from the California Institute for Regenerative Medicine to develop this project to the stage where the first clinical trials can be engaged into. In the first year, we had fully validated the stem cell population of use on a large number of human adipose samples and clearly documented the availability, efficiency and safety of these cells independently of the age, sex and weight of the donor. We had also initiated experiments in mice and rats which have been pursued during the second year, finally demonstrating unequivocally the outstanding bone forming ability of these cells in the presence of the appropriate scaffold and osteogenic hormone. We have also, during this 2nd year, developed a large animal model of stem cell mediated bone regeneration which will be indispensable prior to the inception of human treatments. Finally, we have refined the physicochemical properties of the scaffold for optimal osteogenic factor availability and bone growth. In conclusion, all milestones and deadlines included in this grant have been met and our team is steadily progressing toward the medical “translation” of these novel stem cells.
  • Our research group has identified in the human body a novel class of stem cells endowed with strong osteogenic potential, which means capacity to produce bone. These stem cells are attached to the outer aspect of blood vessels, and therefore present in all organs. Our ultimate goal is to use these stem cells in human beings, to perform for instance spine fusion, a procedure used to treat spine deformities (scoliosis) or faulty vertebrae, or to heal complicated bone fractures, especially in patients suffering from osteoporosis, a condition where spontaneous bone repair is compromised. We shall harvest stem cells from the patient’s own abdominal adipose (fat) tissue, a rich source of these cells which is also easy and safe to collect. Stem cells will be purified on an automated machine, named fluorescence activated cell sorter, and immobilized on an engineered biocompatible scaffold in the presence of a novel osteogenic growth factor, that is a sort of hormone which stimulates stem cell development into bone. We have won an Early Translational grant (2011-2014) from the California Institute for Regenerative Medicine to develop this project to the stage where the first clinical trials can be engaged into. In the first year, we had fully validated the stem cell population of use on a large number of human adipose samples and clearly documented the availability, efficiency and safety of these cells independently of the age, sex and weight of the donor. We had also initiated experiments in mice and rats which have been pursued during the second year, finally demonstrating unequivocally the outstanding bone forming ability of these cells in the presence of the appropriate scaffold and osteogenic hormone. We have also, during this 2nd year, developed a large animal model of stem cell mediated bone regeneration which will be indispensable prior to the inception of human treatments. Finally, we have refined the physicochemical properties of the scaffold for optimal osteogenic factor availability and bone growth. In conclusion, all milestones and deadlines included in this grant have been met and our team is steadily progressing toward the medical “translation” of these novel stem cells.

Enhancing healing via Wnt-protein mediated activation of endogenous stem cells

Funding Type: 
Early Translational I
Grant Number: 
TR1-01249
ICOC Funds Committed: 
$6 762 954
Disease Focus: 
Bone or Cartilage Disease
Stroke
Neurological Disorders
Heart Disease
Neurological Disorders
Skin Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration. Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic. Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries. The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.
Statement of Benefit to California: 
Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B. We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.
Progress Report: 
  • In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
  • Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
  • In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
  • Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
  • In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
  • We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
  • In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.
  • The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.
  • To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.
  • This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.
  • At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.
  • In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.
  • In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.
  • This progress report covers the period between Sep 01 2013 through April 30 2014, and summarizes the work accomplished under ET funding TR101249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for purposes of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians. At the conclusion of Year 2 an external review panel was assembled and charged with the selection of a single lead indication for further development. The scientific advisory board unanimously selected skeletal repair as the lead indication.
  • In year 3 we accrued addition scientific evidence, using both large and small animal models, demonstrating that a WNT protein therapeutic accelerated bone healing. Also, we developed new methods to streamline the purification of WNT proteins, and improved our method of packaging of the WNT protein into liposomal vesicles (e.g., L-WNT3A) for in vivo use.
  • In year 4 we clarified the mechanism of action of L-WNT3A, by demonstrating that it activates endogenous stem cells and therefore leads to accelerated bone healing. We also continued our development studies, by identifying a therapeutic dose range for L-WNT3A, as well as a route and method of delivery that is both effective and safe. We initiated preliminary safety studies to identify potential risks, and compared the effects of L-WNT3A with other, commercially available bone growth factors.
  • In year 5 we initiated two new preclinical studies aimed at demonstrating the disease-modifying activity of L-WNT3A in spinal fusion and osteonecrosis. These two new indications were chosen by a CIRM review panel because they represent an unmet need in California and the nation. We also initiated development of a scalable manufacturing and formulation process for both the WNT3A protein and L-WNT3A formulation. These two milestones were emphasized by the CIRM review panel to represent major challenges to commercialization of L-WNT3A; consequently, accomplishment of these milestones is a critical yardstick by which progress towards an IND filing can be assessed.

Skeletogenic Neural Crest Cells in Embryonic Development and Adult Regeneration of the Jaw

Funding Type: 
New Faculty II
Grant Number: 
RN2-00916
ICOC Funds Committed: 
$2 396 871
Disease Focus: 
Bone or Cartilage Disease
Trauma
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The goal of this proposal is to develop cell-based therapies that lead to the better healing of traumatic head injuries. Our first strategy will be to use genetics and embryology in zebrafish to identify factors that can convert human embryonic stem cells into replacement skeleton for the head and face. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. As zebrafish develop rapidly and can be grown in large numbers, a growing number of researchers are using zebrafish to study how and when cells decide to make a specific type of tissue – such as muscle, neurons, and skeleton - in the vertebrate embryo. Recently, we have isolated two new zebrafish mutants that completely lack the head skeleton. By studying these mutants, we hope to identify the cellular origins and genes that make head skeletal precursors in the embryo. These genes will then be tested for their ability to drive human embryonic stem cells along a head skeletal lineage. Our second strategy will be to test whether a population of cells, similar to the one that makes the head skeleton in the embryo, exists in the adult face. We have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation. In this proposal, we use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can give rise to new head skeleton in response to injury. Traumatic injuries to the face are common, and treatment typically involves grafting skeleton from other parts of the patient to the injury site. Unfortunately, the amount of skeleton available for grafts is in short supply, and surgeries often result in facial disfigurement that causes psychological suffering for the patient for years to come. Here we propose two better treatments that would lead to more efficient healing and less scarring. The first treatment would be to differentiate human embryonic stem cells, a potentially limitless resource, into skeletal precursors that can be grafted into the head injury site. By understanding the common pathways by which head skeletal cells are specified in the zebrafish embryo and human embryonic stem cells, we will be able to generate skeletal replacement cells in large quantities in cell culture. The second treatment would be to stimulate adult stem cells already in the face to regenerate the injured head skeleton. If successful, our experiments on zebrafish jaw regeneration will allow us to devise strategies to augment the natural skeletal repair mechanisms of humans.
Statement of Benefit to California: 
Traumatic injuries to the head, such as those caused by car accidents and gunshot wounds, are commonly seen in emergency rooms throughout California. Current treatments for severe injuries of the head skeleton involve either grafting skeleton from another part of the body to the injury site or, in cases where there is not sufficient skeleton available for grafts, implanting metal plates. Although these operations save lives, they often result in facial disfigurement that causes psychological suffering for the patient for years to come. For this reason, there is enormous interest in cell-based skeletal replacement therapies that will heal the face without leaving disfiguring scars. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. Thus, we are using the zebrafish embryo to rapidly identify factors that can make head skeletal precursors, and then asking if these same factors can instruct human embryonic stem cells to form skeletal replacement cells. In addition, we have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation, and we will use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can regenerate the face. The successful completion of these experiments would allow us to both generate unlimited amounts of head skeletal precursors for facial repair and stimulate latent skeletal repair mechanisms. The combination of these approaches will lead to therapies that promote a more natural healing of the face, thus allowing Californians to eventually resume normal lives after catastrophic accidents.
Progress Report: 
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.
  • In the first year of this award, we have made a landmark discovery concerning how the skeletal-forming cells arise in the head. The head skeleton arises from an unusual cell population called the neural crest, which is characterized by its ability to form a very wide diversity of cell types in the embryo. We had previously described the isolation of two mutant lines of zebrafish that completely and specifically lack the head skeleton. We have now identified a mutation in a variant histone protein as the genetic basis for the lack of head skeleton in one of these lines. Histone proteins play a central role in not only wrapping our DNA but also controlling which genes are active in different cell types. Despite the fact that histones are expressed in every cell in the body, we have found that variant histones are uniquely required for neural crest cells to acquire skeleton-forming potential. In particular, our findings indicate that the head skeleton forms by a process of developmental reprogramming, in which precursor cells with a limited potential undergo large-scale changes in their DNA packaging such that they are able to form a much wider array of cell types.
  • Controlled cell reprogramming is becoming one of the most promising directions of regenerative medicine. For example, there is enormous therapeutic potential in being able to take cells from a patient, reprogram these cells back to a naïve state with defined factors, and then induce these cells to form replacement cells of any type that can be introduced back into the patient. Our discovery that the vertebrate embryo uses a similar process of reprogramming to generate head skeletal cells during development provides us an opportunity to better understand how reprogramming works at a molecular level. In addition, our finding that head skeletal cells form by developmental reprogramming suggests that adult patient-specific cells can be directly reprogrammed to form head skeletal precursors. Moreover, as the variant histone we have identified is identical at the protein level between zebrafish and humans, it is likely that the reprogramming process we have discovered operates in humans as well. Thus, in the coming years of this grant we are excited to develop methods of reprogramming adult patient-specific cells to a head skeletal fate, such that we can generate large quantities of replacement cells to repair the face and skull.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head. An understanding of how these skeletal cells form in the embryo will aid in our long-term goal of producing skeletal replacement cells in culture and stimulating new skeleton to form after traumatic head injuries.
  • In the second year of this award, we have extended our initial findings that variant histones play an essential role in the generation of head skeletal cells. Skeletal cells usually arise from a cell population called the mesoderm. However, in the head a unique population of ectoderm cells, which normally forms derivatives such as neurons and skin, has an added ability to form skeletal cells. How these cranial neural crest cells acquire this extra potential remains unclear. Here we show that variant histones function within the ectoderm to give cranial neural crest cells skeletal-forming ability. In addition, we have used biochemical analysis in a human cell line to demonstrate how mutations in a particular H3.3 type of variant histone disrupt the association of histones with DNA. Furthermore, we have developed tools which allow us to analyze how variant histone changes throughout the genome endow neural crest ectoderm cells with mesoderm-like skeletal-forming potential.
  • Variant histones have been implicated in cell reprogramming, whereby a mature cell regains the ability to form many more cells that can repair a damaged tissue. As we find that variant histones are required for reprogramming of the ectoderm to form neural crest during early development, we believe that we can use this pathway to convert patient-specific cells to neural crest and skeletal cells that can be used for facial repair. To test this, we have developed a mouse model in which we can detect the ability of neural crest genes to convert mature cells to neural crest and skeletal fates. In parallel, we have developed a model of jaw regeneration in zebrafish that will allow us to test whether adult cells within the animal can be reprogrammed to repair the skeleton in response to injury. During this period, the generation of transgenic tools has allowed us to begin to address which cell types can give rise to new skeleton in response to injury. In the coming years, we hope that our work in model systems will lead to therapies for head skeletal injuries on two fronts: the generation of large amount of head skeletal precursors from patient-specific cells and the induction of increased regenerative ability of cells within the patient.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We have previously described that zebrafish with a mutant form of a variant histone H3.3 protein have very specific defects in the ability of neural crest cells to form skeleton. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In the third year of this award, we have expanded our analysis to examine how the incorporation of histone H3.3 is regulated specifically in the neural crest population. Our data suggest that such H3.3 incorporation may depend on a novel chaperone protein as reducing the function of several known H3.3 chaperone proteins does not lead to specific neural crest or head skeletal defects. A clue to what regulates H3.3 activity comes from a second zebrafish mutant – called myx - that we are studying, which has head skeletal defects similar to what we see in our H3.3 mutant. We have mapped the myx mutant interval to a very small region that contains a putative H3.3 chaperone protein. We are currently establishing whether loss-of-function of this chaperone accounts for myx defects. Together, our studies of H3.3 and myx mutants will shed light on how to generate cells with the ability to form replacement head skeleton in patients. To this aim, we have also begun experiments to use our findings in zebrafish to directly convert mammalian cells (initially mouse but then in humans) to a neural crest and skeletal fate.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that the zebrafish lower jaw bone regenerates through a cartilage intermediate, in contrast to the direct differentiation to bone during development. Hence, our findings indicate that bone regeneration in zebrafish is a cellularly distinct mechanism than bone development. Furthermore, we have found that the FGF signaling pathway is greatly upregulated during early jaw regeneration. FGF signaling also mediates the regeneration of the heart and other organs in zebrafish, and thus jaw regeneration may rely on a common regenerative program throughout the zebrafish. In the coming period, we plan to test the functional requirements of FGF signaling in mediating jaw regeneration, as well as identifying the stem cell populations that are the FGF-dependent source of new bone.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. In the previous grant cycle, we published a manuscript in PLoS Genetics describing the role of a variant histone H3.3 protein in controlling the ability of neural crest cells to form the head skeleton of zebrafish. As histone H3.3 is a core component of the chromatin around which DNA is wrapped, our findings suggest a novel mechanism by which changes in chromatin structure endow the neural crest with the ability to form a wide array of derivatives. In addition, we published a separate study in PLoS Genetics that showed a critical role of Twist1 in guiding these neural crest cells to make head skeleton at the expense of other cell types such as neurons. Together, our studies of H3.3 and Twist1 in zebrafish will shed light on how to generate cells with the ability to form replacement head skeleton in patients. In ongoing experiments, we are using principles from our zebrafish system to directly convert mammalian cells (initially in mouse but then in humans) to a neural crest and skeletal fate.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. We have a limited ability to repair defects in our skeleton, for example after bone fracture. However, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jaw following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, an unusual cartilage intermediate is able to directly make replacement bone, which is in marked contrast to the way bone is made during development. Furthermore, we have found a potentially critical role of the Ihh signaling pathway in allowing regenerating cartilage cells to directly make replacement bone. In the coming period, we plan to test the functional requirements of Ihh signaling in mediating jaw regeneration, as well as identifying the cellular source of bone-producing cartilage cells during jaw regeneration. As similar bone-producing cartilage cells may also be present in human fractures, lessons learned from zebrafish may allow us to stimulate these cells and hence augment bone repair in patients.
  • A major aim of this grant is to investigate the developmental origin of the skeleton-forming cells in the head, as well as their ability to regenerate craniofacial skeleton in adults after injury. The head skeleton derives from a special population of cells, the neural crest, which has the remarkable ability to form not only neurons but also skeletal tissues. We had previously identified a unique role of histone replacement within the neural plate precursor cells that allows neural crest to make skeletal derivatives. In the current grant cycle, we now find that misexpression of groups of neural plate transcription factors is able to convert cells to a neural crest fate, and we are currently exploring whether this occurs by stimulating the histone replacement program we found to be so important for neural crest development. We are also testing whether similar neural plate transcription factors can convert mammalian cells to a neural crest fate, with the eventual goal to use this technique to generate an unlimited supply of patient-specific bone and cartilage replacement cells for skeletal repair.
  • A parallel strategy that we are taking towards regenerative strategies for facial skeleton is to stimulate endogenous neural crest cells to make replacement skeleton. While we have a limited ability to repair defects in our skeleton, for example after bone fracture, we have found that adult zebrafish have the remarkable ability to regenerate nearly their entire lower jawbone following amputation. By studying why zebrafish regenerate facial skeleton to a much greater extent than humans, we hope to devise molecular strategies to augment skeletal repair/regeneration in patients. In particular, we have found that during zebrafish lower jawbone regeneration, cartilage cells are able to change their fate to directly make replacement bone, which is in marked contrast to the way bone is made during development. We have also identified a critical role of the Ihh signaling pathway in bone regeneration and in particular the generation of the critical cartilage intermediate. In adults lacking the Ihha protein, no cartilage forms after jawbone amputation and the jawbone fails to heal properly. Moreover, in collaborative work we find that such bone-producing cartilage cells may also be present in a mammalian model of bone healing. We are therefore excited by the prospects of using similar bone-producing cartilage cells to repair large skeletal wounds in patients.

Treatment of osteoporosis with endogenous Mesenchymal stem cells

Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05302
ICOC Funds Committed: 
$19 999 867
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Although most individuals are aware that osteoporosis is disease of increased bone fragility that results from estrogen deficiency and aging, most are unaware of the high risk and cost of the disorder. It is estimated that close to 30% of the fractures that occur in the United States each year are due to osteoporosis (Schwartz & Kagan 2002). California, with one of the largest over-age-65 populations, is expected to double the fracture rate from 1995 to 2015 (Schwartz & Kagan 2002). Current treatment of osteoporosis is focused on anti-resorptive agents that prevent further bone loss. These agents and are effective in reducing new vertebral fractures but less effective for the prevention of hip fractures, and the duration of use of one anti-resorptive class, the bisphosphonates, is limited due to a concern about weakening of the cortical bone with longterm use. The only bone growing agent that is approved by FDA is the protein, hPTH 1-34, which requires two years of daily injections, is only approved by the FDA for one course of treatment, is only effective in about 60% of treated individuals for reduction of vertebral fractures, and has not been shown to be effective in reducing new hip fractures. This leaves an unmet medical need for an anabolic or agent that stimulates bone formation for millions of elderly Californians that suffer or will suffer from this disease. We have developed a small molecule, LLP2A-Ale that directs endogenous mesenchymal stem cells (MSCs), the cells that have the potential to grow bone tissue, to the bone surface to form new bone. We propose a development plan for this small molecule, LLP2A-Ale for the treatment of osteoporosis in both postmenopausal women and men. Yrs. 1-2: These 2 years will be spent with optimizing the manufacturing and packaging of the small molecule, obtaining information about the efficacy and toxicity in preclinical models, and preparing documents for an FDA meeting when the preclinical studies are completed to provide comment on the proposed Phase I clinical trials. Yrs. 3-4. We plan perform a Phase I study with two parts. Part I will study postmenopausal women with osteopenia and a fracture risk (3% for hip fracture and 20% for major nonvertebral fractures over the next 10 years). After the initial Phase I study in postmenopausal women we will perform Part 2 and study both postmenopausal women and men with similar inclusion and exclusion criteria. The primary endpoint of these studies will be change in biochemical markers of bone turnover (PINP, BSAP, osteocalcin), and secondary endpoints will be bone mineral density of the lumbar spine measured by DXA and trabecular bone volume measured by QCT. The Phase I trials will also include required pharmacokinetic and pharmacodynamic measures to obtain information about the action of this small molecule and to inform us for Phase II clinical studies in the future.
Statement of Benefit to California: 
Osteoporosis is a disease of the elderly that results from a process of age related bone loss that renders the bone fragile. Current osteoporosis treatments have relatively good efficacy in reducing incident fractures. However these agents (anti-resorptive agents or the anabolic agent rhPTH (1-34) only reduce the risk of vertebral fractures about 60%, and hip fractures only 40%, and these agents require years of treatment to be effective. The goal of this project is to increase bone homing of the endogenous MSCs with a small molecule (LLP2A-Ale) to form new bone as a novel treatment for osteoporosis that could cure osteoporosis with only 3-4 injections by mobilizing the endogenous MSCs to build bone. Our molecule would be highly competitive in this market as the efficacy of increasing bone mass and bone strength would be high and the risks in a very acceptable range. The market potential for bone tissue regeneration is large as it is estimated that close to 1/3 of fractures that occur in the US each year are due to osteoporosis (Schwartz & Kagan (2002). California, with one of the largest over-age-65 populations, is expected to double the fracture rate from 1995 to 2015 (Schwartz & Kagan 2002). One study places the cost per year in osteoporotic fractures at 2.4 billion dollars (Schwartz & Kagan 2002), establishing it as one of the highest health care costs for older individuals. The prevalence of osteoporosis is projected to increase with increasing lifespan globally both from age related bone loss and from secondary causes of bone loss including inflammatory diseases and cancer. The market potential for bone tissue regeneration is large, an estimated 2 million fractures and $19 billion in costs annually. By 2025, experts predict that osteoporosis will be responsible for approximately 3 million fractures and $25.3 billion in costs each year (publication from National Osteoporosis Foundation). The osteoporotic patients spend about $10 a month for the generic version of Fosamax, at the lower end, to about $80 a month for brand-name Fosamax or Actonel to $900 or more a month for Forteo (rhPTH (1-34). Therefore, once validated in osteoporosis patients, this form of tissue regeneration would be effective in patients with primary osteoporosis, in patients with secondary osteoporosis due to long term glucocorticoid treatment or after chemotherapy in both men and women and to augment peak bone mass in children in whom current osteoporosis medications are contraindicated, in individuals who have had radiation to their skeletons in whom rhPTH (1-34) is contraindicated and to augment fracture healing in the elderly. Our agent would have the potential to save the State of California millions of dollars in health care and would allow these osteoporotic individuals to live longer and be independent longer.
Progress Report: 
  • One of the early goals for this project is the successful development and clinical grade manufacturing of the drug LLP2A-Alendronate (LLP2A-Ale). We are pleased that we now have a robust stability indicating method that has been transferred for use in drug product development. We are currently working with our collaborators on stability maintenance and monitoring of the compound.
  • At the annual CIRM advisory committee review in late October 2013, the reviewers liked the "hybrid" compound. However, they also felt that the project would benefit from additional preclinical studies to compare two treatments for osteoporosis that are currently available, alendronate and PTH. Therefore, based on the advisory committee's comments, we will conduct further studies that confirm and support our original hypotheses. We are looking forward to beginning clinical trials soon after those studies are completed.

Increasing the endogenous mesenchymal stem cells to the bone surface to treat osteoporosis

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05302
ICOC Funds Committed: 
$110 000
Disease Focus: 
Bone or Cartilage Disease
oldStatus: 
Closed
Public Abstract: 
Although most individuals are aware that osteoporosis is disease of increased bone fragility that results from estrogen deficiency and aging, most are unaware of the high risk and cost of the disorder. It is estimated that close to 30% of the fractures that occur in the United States each year are due to osteoporosis (Schwartz & Kagan (2002). California, with one of the largest over-age-65 populations, is expected to double the fracture rate from 1995 to 2015 (Schwartz & Kagan 2002). One study places the cost per year in osteoporotic fractures at 2.4 billion dollars (Schwartz & Kagan 2002), establishing it as one of the highest health care costs for older individuals. The prevalence of osteoporosis is projected to increase with increasing lifespan globally both from age related bone loss and from secondary causes of bone loss including inflammatory diseases and cancer. In additional, medications used for the treatment of cancer and inflammatory diseases can also induce bone loss. Current treatment of osteoporosis is focused on agents that prevent further bone loss such as the bisphosphonates or selective estrogen modulators. The only bone growing agent that is approved by FDA is the protein, hPTH 1-34, which requires two years of daily injections and is only effective in about 60% of treated individuals. We have developed a small molecule, LLP2A-Alendronate that augments the homing of endogenous mesenchymal stem cells (MSCs), the cells that have the potential to grow bone tissue, to the bone surface and form new bone. Therefore, we plan to file IND in the next sin months and we will perform two clinical trials to test its safety and efficacy in two clinical trials in the next fours years. Yrs. 1-2: Phase I clinical trial. To determine if LLP2A-Ale is safe when used in patients with osteoporosis. After this phase I study, our research group will decide on two or three doses of LLP2A-Ale and two dosing regimens and will perform a phase II clinical trial. Yrs. 3-4: Our phase II clinical trials will evaluate the efficacy of LLP2A-Ale in patients with osteoporosis The primary endpoint will be bone mineral density measured by DEXA of the lumbar spine and hip and biochemical markers of bone turnover, also calciotrophic hormones of bone metabolism (Vitamin D, FGF23, Sclerostin, IGF-1,and sclerostin,etc). Secondary clinical study endpoints will include a detailed assessment of the quantity of new bone formed and its distribution throughout the skeleton with XtremeCT, a new high-resolution 3 dimensional bone scan that allows regular follow-up measurements with software that automatically matches cortical and trabecular bone regions (SCANCO Medical microCT Systems) at 3 month intervals and bone biopsies performed at the iliac crest after treatment is completed. All the patients in the trials will be followed at 3 month intervals for 2 years.
Statement of Benefit to California: 
Osteoporosis is a disease of the elderly that results from a process of age related bone loss that renders the bone fragile such that it breaks with very little force. Current osteoporosis treatments have relatively good efficacy in improving bone strength and reducing incident fractures, however these agents ( anti-resorptive agents or the anabolic agent rhPTH (1-34) only reduce the risk of hip fractures by 40%, and require years of treatment to be effective. The goal of this project is to increase bone homing of the endogenous MSCs with a novel compound to form new bone as a novel treatment for osteoporosis. A compound that could cure osteoporosis with only 3-4 injections of an agent that mobilized MSCs to build bone would be highly competitive in this market as the efficacy of increasing bone mass and bone strength would be high and the risks in a very acceptable range. This agent would be effective in patients with primary osteoporosis defined by very low bone mass or low trauma fractures, in patients with secondary osteoporosis due to long term glucocorticoid treatment or after chemotherapy in both men and women and to augment peak bone mass in adolescents. The market potential for bone tissue regeneration is large, an estimated two million fractures and $19 billion in costs annually. By 2025, experts predict that osteoporosis will be responsible for approximately three million fractures and $25.3 billion in costs each year (publication from National Osteoporosis Function). The osteoporotic patients spend about $10 a month for the generic version of Fosamax, at the lower end, to about $80 a month for brand-name Fosamax or Actonel to $900 or more a month for Forteo (huPTH (1-34).A compound that could cure osteoporosis with only 3-4 injections of an agent that mobilized MSCs to build bone would be highly competitive in this market as the efficacy of increasing bone mass and bone strength would be high and the risks in a very acceptable range. Once validated in osteoporosis patients, this form of tissue regeneration will be useful for children in whom current osteoporosis medications is contraindicated, individuals who have had radiation to their skeletons, and to augment fracture healing in the elderly. The market potential for bone tissue regeneration is large as it is estimated that close to 1/3 of fractures that occur in the US each year are due to osteoporosis (Schwartz & Kagan (2002). California, with one of the largest over-age-65 populations, is expected to double the fracture rate from 1995 to 2015 (Schwartz & Kagan 2002). One study places the cost per year in osteoporotic fractures at 2.4 billion dollars (Schwartz & Kagan 2002), establishing it as one of the highest health care costs for older individuals. The prevalence of osteoporosis is projected to increase with increasing lifespan globally both from age related bone loss and from secondary causes of bone loss including inflammatory diseases and cancer.
Progress Report: 
  • The purpose of our Disease Team planning grant was to develop a solid Clinical development program for our compound, LLP2A- Ale that directs mesenchymal stem cells to the bone marrow and stimulates new bone formation.
  • We published our scientific findings in the Journal Nature Medicine (2012 March issue). Our research team developed a clinical development program that included producing compound, performing toxicity studies, and a clinical development plan for a Phase I study. We also submitted an IND for the FDA, the FDA responded to our clinical development program and questions we raised, and we had a teleconference with the FDA CDER group on January 31st, 2012.
  • We have obtained sufficient information from the FDA on our IND to begin producing our compound for toxicity studies. In addition, we participated in a Stem Cell Awareness Public Forum at UC Davis in which we displayed our work with the mesenchymal stem cells to build new bone.

Clinical Development of an osteoinductive therapy to prevent osteoporosis-related fractures

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05368
Investigator: 
ICOC Funds Committed: 
$99 110
Disease Focus: 
Bone or Cartilage Disease
oldStatus: 
Closed
Public Abstract: 
There are over 1.5 million osteoporotic fractures annually in the USA alone, at a cost of approximately $15 billion each year. The majority of these fractures occur in the spine, followed by the hip and wrist. Incidence varies according to age; vertebral fracture rates increase rapidly by the sixth decade of life, whereas the risk of hip fracture rises markedly by the eighth decade and beyond. Current treatment is focused on prevention using osteoclast inhibitors, hormone therapy, diet and exercise. When a fracture occurs current therapies involve injection of cement into the vertebral body and/or open surgery with implants. Unfortunately, these procedures do not regenerate bone tissue, often fail and incur risks of leakage and emboli. The clinical and economic impact associated with these fractures is substantial. Following a fragility fracture, significant pain, disability, and deformity can ensue. If fracture union is not achieved, the patient may suffer long-term disability. This is exacerbated because there is a five-fold increase in the risk for sustaining a subsequent vertebral fracture and the odds that a neighboring vertebrae will fail within one year is >20%. We propose to add a noninvasive anabolic option to the treatment and prevention of osteoporotic fractures. This therapy utilizes a novel small molecule Wnt pathway activator that drives the endogenous stem cells in the bone compartment to differentiate into bone forming osteoblasts thereby increasing bone mass and reducing the risk of fracture. This therapy will be administered 1-2X/year by injection, eliminating the concerns over patient compliance and revolutionizing the treatment of vertebral and hip fractures in patients suffering from osteoporosis.
Statement of Benefit to California: 
There are over 25 million osteoporosis patients in the US alone, leading to 1.5 million osteoporotic fractures annually at a cost of approximately $17 billion per year. The lifetime incidence of fragility fractures secondary to osteoporosis in females over fifty years of age is approximately 1 in 2, and in males over the age of fifty, is 1 in 4. Osteoporosis-related vertebral compression fractures are the most common fragility fractures in the United States, accounting for more than 79% of the total. Approximately 70,000 OVCFs result in hospitalization each year with an average hospital stay per patient of 8 days. Current treatment is focused on prevention using osteoclast inhibitors, hormone therapy, diet and exercise. When a fracture occurs, current therapies involve injection of cement into the vertebral body and/or open surgery with implants. Unfortunately, these procedures do not regenerate bone tissue, often fail, incur risks of leakage and emboli, and suffer significant side effects. The clinical and economic impact associated with these fractures is substantial. Following a fragility fracture, significant pain, disability, and deformity can ensue. If fracture union is not achieved, the patient may suffer long-term disability. This is exacerbated because there is a five-fold increase in the risk for sustaining a subsequent vertebral fracture after the first fracture, and the odds that an adjacent vertebrae will fail within one year is >20%. We propose to add a noninvasive anabolic option to the treatment and prevention of osteoporotic fractures, with minimal to no side effects or systemic safety concerns. This therapy utilizes a novel small molecule Wnt pathway activator that drives the endogenous stem cells in the bone compartment to differentiate into bone forming cells, thereby increasing bone mass and reducing the risk of fracture. This therapy will be administered 1-4 times per year by injection, eliminating the concerns over patient compliance and revolutionizing the treatment of vertebral and hip fractures in patients suffering from osteoporosis. This will benefit the citizens of California by reducing hospitalization periods, operative costs and loss of workdays, and by improving quality of life for Californians with osteoporosis that are at risk for OVCFs.
Progress Report: 
  • This project is working to advance a first-in-class, small molecule Wnt pathway activator through IND-enabling and Phase I/II clinical studies for treatment of osteoporotic hip fractures. During this reporting period, quotes were solicited from drug manufacturing companies for the manufacture of the drug compound, a manufacturer was selected and the drug is presently in the GMP manufacturing process. In addition, international thought leaders were identified to act as clinical consultants and with their input the outline of a Phase I/II clinical trial plan was drafted and submitted to the FDA for comment. Beyond that, a pre-IND briefing document was prepared that described the clinical product’s known pharmacology, pharmacokinetic, toxicology and chemical characteristics. After preparing and submitting the clinical plan outline and pre-IND briefing document to the FDA, the team had a successful pre-IND meeting on January 30, 2012. IND filing is proposed for the first half of 2013. Currently, quotes have been requested for contract research organizations (CROs) who offer appropriate preclinical fracture model studies and a decision on the most suitable vendor for the study will be determined shortly.
  • In addition, the CIRM disease team application detailing known properties of the drug, along with a 4 year development plan and associated budget was drafted and completed during the reporting period.

Genetically Engineered Mesenchymal Stem Cells for the Treatment of Vertebral Compression Fractures.

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05288
ICOC Funds Committed: 
$109 743
Disease Focus: 
Bone or Cartilage Disease
oldStatus: 
Closed
Public Abstract: 
Osteoporosis is an unsolved and highly prevalent health care problem: 10 million Americans suffer from the disease, and an additional 34 million have low bone mass. Roughly half of all women and a fourth of all men older than 50 years will sustain an osteoporosis-related fracture at some time in their lives, and when such a fracture occurs, the chances of death within 12 months are about 1 in 5. Osteoporotic fractures can take several forms, but VCFs (vertebral compression fractures) occur at a rate of 700,000 per year—twice the rate of hip fractures. The economic burden of osteoporotic fractures is tremendous. In 2001, there were approximately 1.5 million osteoporosis-related fractures in the US at a cost of $17 billion, or approximately $47 million per day. Currently, treatment is focused primarily on prevention. When fractures occur in patients with osteoporosis, treatment options are limited because open surgery with implants often fails. Recently, new therapies involving injection of cement into the vertebral body were developed. Unfortunately, these procedures do not regenerate bone tissue, but do incur risks of leakage and emboli. Moreover,recent publications in leading scientific journal question the effectiveness of those procedures. Hence, we need new biological treatment that will promote repair of such fractures in a safe and efficient manner. We propose to develop a therapy that exploits MSCs (mesenchymal stem cells) that are genetically engineered to express a bone-inducing gene, bone morphogenetic protein-2 (BMP-2). Those cells have been shown to induce bone formation and fracture repair in numerous studies in animal models. Specifically, we intend to use allogeneic ("off the shelf") human MSCs. These cells will be genetically engineered with a BMP-2 gene using a technology based on short electric pulses. BMP-2 engineered MSCs have an advantage in bone repair since they become bone cells by themselves and recruit additional cells from the environment. This synergistic effect leads to accelerated and robust bone formation, which could be an attractive therapy for a variety of clinical conditions involving bone lose. An image-guided injection of BMP-2 engineered MSCs to a fractured vertebra could be an attractive treatment that would lead to rapid fracture repair and shortening of hospitalization time. We propose to use off-the-shelf MSCs that do not require the patient to undergo additional medical procedure such as bone marrow aspiration. In addition, the use of allogeneic cells is not limited by cell number, as could be the case for autologous cells, that are taken from the patient. If successful, this therapeutic strategy could revolutionize the treatment in osteoporsis patients, offering a minimal-invasive, biological solution. We plan to analyze aspects of efficiency and safety of the proposed therapy in a pre-clinical model, that will enable us to submit an approvable IND to the FDA by the end of the 4-year project.
Statement of Benefit to California: 
Approximately 10 million people in the United States are diagnosed as osteoporotic, while an additional 34 million are classified as having low bone mass. The lifetime incidence of fragility fractures secondary to osteoporosis in females over fifty years of age is approximately 1 in 2, and in males over the age of fifty, is 1 in 4. Vertebral compression fractures (VCFs) are the most common fragility fractures in the United States, accounting for approximately 700,000 injuries per year, twice the rate of hip fractures. Approximately 70,000 VCFs result in hospitalization each year with an average hospital stay per patient of 8 days. Fragility fractures due to osteoporosis also place a severe financial strain upon the health care industry. Estimates show there were approximately 1.5 million osteoporosis-related fractures in the United States in 2001, the care of which cost about $17 billion. Moreover, as the number of individuals over the age of fifty continues to increase, costs are predicted to rise to an estimated $60 billion a year by the year 2030. VCFs have previously received limited attention from the spine care community. This oversight may be a result of the perception that VCFs are benign, self-limited problems or that treatment options are limited. However, it has become clear that VCFs are associated with significant physiologic and functional impairment, even in patients not presenting for medical evaluation at the time of fracture. Current treatment of osteoporotic patients is mostly focused on prevention of VCFs. There are a few options of treatment when VCFs actually occur. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, nonoperative management, including medications and bracing, is usually recommended for the vast majority of patients. Unfortunately, large numbers of patients report intractable pain and inability to return to activities. Currently there is no efficient biological solution for the treatment of VCFs. The proposed study will further develop a biological therapeutic solution that will accelerate repair of VCFs. The treatment will rely upon adult stem cell that are genetically engineered to overexpress an osteogenic gene, BMP-2, using a non-viral technique that is currently in clinical trials. It will also involve an injection of the cells into the fracture site, instead of a percutaneous injection of a polymer, which does not restore lost bone tissue. Data generated form this study could potentially revolutionize the treatment of vertebral fractures and other complex fractures in patients suffering from osteoporosis, and so benefit the citizens of California by reducing hospitalization periods, operative costs and loss of workdays, and by improving quality of life for Californians with osteoporosis that are at risk for VCFs.
Progress Report: 
  • Approximately 10 million people in the United States are diagnosed as osteoporotic, while an additional 34 million are classified as having low bone mass. The lifetime incidence of fragility fractures secondary to osteoporosis in females over the age of fifty years of age is approximately 1 in 2, and in males over the age of fifty, is 1 in 4. Vertebral compression fractures (VCFs) are the most common fragility fractures in the United States, accounting for approximately 700,000 injuries per year, twice the rate of hip fractures. Approximately 70,000 VCFs result in hospitalization each year with an average hospital stay per patient of 8 days. There are limited options of treatment when VCFs occur. New, non-biological, methods have been developed to regain the biomechanical properties of a fractured vertebral body. These methods include the minimally invasive procedures of vertebroplasty and balloon tamp reduction. Both procedures involve injection of synthetic nonbiological material that does not resorb and instead remains a permanent foreign-body fixture in the spine. Ultimately, a biological solution that would promote rapid fracture healing and stimulate normal bone production would be the best for osteoporotic patients with vertebral column injuries. Our goal is to develop a novel therapy for VCFs, which will be based on adult stem cells, isolated from bone marrow samples. These cells will be "triggered" to form bone by the activation of a specific gene and then injected into the fractured vertebra leading to fast fracture repair. The funding from the planning award allowed us to establish a highly experienced team, from the academy and industry, that would be able to bring the proposed therapy to clinical use. In addition, we assembled a comprehensive development plan that described in detail the studies and steps required to obtain an approval from the FDA in order to begin clinical trials. Furthermore, we have negotiated and obtained commitment from several biotech companies, which will provide the necessary materials and facilities to perform the studies described in the development plan. Our proposal for a Disease Team Therapy Development Award was submitted in January 2012. If awarded, we will be beginning the pre - clinical studies in August 2012.

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