“Next-generation” mesenchymal stem or stromal cells for the in vivo delivery of bioactive factors: progressing toward the clinic

Mesenchymal stem cells or marrow stromal cells (MSCs) are currently being tested for multiple indications in Phase I to III clinical trials. The use of “next-generation” MSC therapies, involving combination products and/or genetically engineered MSCs to deliver bioactive factors to damaged tissues, is the next frontier. Our teams at the UC Davis Institute for Regenerative Cures are conducting investigational new drug (IND)-enabling biosafety and efficacy studies for two next-generation products: MSCs engineered to secrete vascular endothelial growth factor (MSC/VEGF) for critical limb ischemia (CLI) and MSCs engineered to produce brain-derived neurotrophic factor (MSC/BDNF), designed to treat Huntington’s disease (HD).

MSC THERAPY FOR THERAPEUTIC ANGIOGENESIS

Numerous clinical trials have demonstrated the biosafety of systemic infusion of allogeneic MSCs into patients with various diseases.^1,2 Osiris Therapeutics, Inc., has conducted multiple clinical trials using allogeneic MSCs administered through systemic infusion. Their studies provide extensive safety and provisional efficacy data for nonmatched allogeneic marrow-derived MSC administration to patients through FDA-approved clinical trials.

CLI is characterized by significant arterial occlusive disease of the lower extremity, which often results in limb loss if left untreated. The symptoms associated with this very severe form of peripheral artery disease are pain in the leg and foot at rest, nonhealing ulcers, limb or digital gangrene, and delayed wound healing. It is estimated that 160,000 to 180,000 major and minor amputations are performed annually in the United States for CLI. Long-term survival is significantly affected by major amputation with only 55% of subjects surviving at 3 years after major amputation.

Liew and O’Brien³ reviewed the progress of using MSCs to treat CLI. Early-phase trials for heart and limb revascularization have shown safety and early indication of efficacy (REVASCOR-Mesoblast, RESTORE-CLI-Aastrom, Multistem-Athersys, and others). The lack of infusion-related serious adverse events in these trials demonstrates the safety of MSCs and other expanded adherent marrow-derived cell infusions when performed carefully and according to specific clinical regimens.

Phase I and II trials of VEGF delivered via plasmid and as purified protein injections showed potential clinical benefit in treatment of CLI, but Phase III studies in this indication did not achieve significant efficacy. The failure of these agents to significantly affect therapeutic angiogenesis has been attributed to the mechanisms of VEGF delivery, because expression from plasmids is suboptimal and transient and the VEGF protein has a short half-life.

The rationale for delivering VEGF from MSCs is to extend the duration of local expression of VEGF and thereby promote angiogenesis. Our preclinical studies demonstrate that MSC/VEGF is useful in promoting angiogenesis, because the cells show tropism toward hypoxic sites and deliver high levels of VEGF from the introduced transgene at the sites of ischemia, promoting local reperfusion of the ischemic tissue. MSC/VEGF cells do not persist indefinitely at the site of ischemia. Rather, they can initiate angiogenesis that is then carried on by endogenous mechanisms once tissue perfusion is restored.

We have previously described the immune-deficient mouse model of hind limb ischemia, which is an established assay to detect angiogenic activity for human stem cells to be used in revascularization therapies.^4-7 Using this method we conducted definitive efficacy and dose-finding studies. In experiments carried out in three different immune-deficient mouse strains, human MSC/VEGF showed significant and sustained therapeutic benefit, compared to animals treated with vehicle alone (Beegle et al., manuscript in preparation, 2015). We had a successful pre-IND meeting with the FDA and are currently conducting definitive biosafety studies in support of a planned future clinical trial for CLI, to be led by vascular expert Dr. John Laird at UC Davis. Dr. Laird’s team is currently conducting two other stem or progenitor trials to treat patients with CLI who have few other options. It is hoped that the stem cell therapy can prevent amputation.

ENGINEERED MSCs FOR A NEUROLOGIC DISORDER: HUNTINGTON’S DISEASE

We are also developing a novel therapy for the devastating neurologic disorder Huntington’s disease (HD): implantation of human MSC/BDNF. BDNF levels are reduced in the brains of HD patients. BDNF has been shown in numerous transgenic HD mouse studies to prevent cell death and to stimulate the growth and migration of new neurons in the brain and is thus a lead candidate for neuroprotection in HD. We are using MSCs as delivery vehicles to produce BDNF in the affected areas of the striatum. We are conducting detailed tests of MSC/BDNF in HD mouse models in preparation for a proposed Phase I clinical trial of MSC/BDNF implantation into the brain of HD patients (HD-CELL), with the goal of slowing disease progression.

We have manufactured and tested human MSC/BDNF using standard operating procedures from our UC Davis Good Manufacturing Practices Facility. We have shown that MSC/BDNF produces high levels of BDNF and that a multiplicity of infection of 10 virus particles per cell generates a single intact integrant per cell, on average. These are data critical to the Recombinant DNA Advisory Committee, for whom we had submitted an Appendix M application, and obtained approval to proceed. Recombinant DNA Advisory Committee approval is needed before FDA approval because we are planning a proposed stem cell gene therapy trial.

We conducted double-blinded studies, examining the effects on disease progression of implantation of human MSC/BDNF in two strains of HD transgenic mice: YAC 128 and R6/2. The R6/2 model has the early onset of neurologic dysfunction and affected animals die much earlier than wild-type or YAC 128 mice. For this reason it is a more suitable model of juvenile HD. In the R6/2 model, we have successfully demonstrated that implantation of MSC/BDNF causes an improvement in deficits in open-field exploration, a behavioral/anxiety assay. We have also shown that MSC/BDNF causes increased neurogenesis in the brains of treated mice, an important milestone (Pollock et al., manuscript in submission, 2015).

The YAC 128 model develops slowly progressive behavior symptoms in midlife and has loss of brain cells that mirrors changes seen in HD patients. In the YAC 128 model, we have shown that implantation of our MSC/BDNF product decreases striatal atrophy between 8 and 12 months of age (Pollock et al., manuscript in submission, 2015). Wild-type mice have a typical lifespan of 2 years, so this age in the YAC 128 mouse roughly corresponds to the typical age at onset for early-stage HD patients that we are proposing to treat in our future planned Phase I study, HD-CELL.

In tandem with the ongoing pre-IND studies in the laboratory, the clinical team is conducting an observational study, PRE-CELL. The goal of PRE-CELL is to establish baseline characteristics and track disease progression in a group of early stage HD patients. PRE-CELL subjects undergo detailed neurologic, psychiatric, cognitive, imaging, and laboratory testing, including measurement of BDNF levels. PRE-CELL participants who have completed at least 1 year of follow-up and meet inclusion and exclusion criteria will be considered for the future planned cell therapy trial. The PRE-CELL trial, led by Dr. Vicki Wheelock at UC Davis, is ongoing (NCT01937923).

We have held a pre-IND meeting with the FDA and are completing additional dose-finding and definitive biosafety studies for the MSC/BDNF product. Our progress to date supports the completion of our final preclinical studies and our plan to go forward toward regulatory approval. There are potential applications of our research beyond HD. Our biologic delivery system for BDNF sets the precedent for adult stem cell therapy in the brain and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, spinocerebellar ataxia, Alzheimer’s disease, and some forms of Parkinson’s disease. It also provides a platform for future gene editing studies.

GENERAL CONSIDERATIONS FOR MSC THERAPEUTICS

Duration of tissue residence is an important factor that we and others are examining. In the acute injury setting, MSCs expanded under standard conditions are only transiently recovered and detectable at low levels at the area of tissue damage at 1 month postinfusion. We have reported that hypoxic preconditioning of the human MSCs enhances their duration of residence and reparative function in an acute tissue ischemia model.^4,8 Implantation into the tissue in a semisolid or injectable biodegradable matrix also improves tissue retention so that the MSCs can express engineered transgenes such as VEGF for a longer period of time, enhancing revascularization effects.

Biosafety is a very important aspect for IND-enabling studies for any proposed trial. We have published a decade-long biosafety study on this aspect of the clinical safety profile of gene-modified MSCs.⁹ The development of cytogenetic abnormalities when MSCs are cultured long term, past the crisis point, can be seen in rodent MSCs, which do not well reflect human MSC biology and are often riddled with phagocytic monocyte-macrophage elements. When genetically engineered human MSCs are cultured under good manufacturing practice or good laboratory practice-like conditions, karyotype is stable and adverse events have not been observed in our rule-out-tumorigenicity studies.

In summary, gene-modified MSCs and MSCs delivered in biodegradable tissue-compatible matrices, the next-generation MSC products, are safe and feasible in animal models and are ready to progress toward the clinic.

ABBREVIATIONS:

CLI

critical limb ischemia

HD

Huntington’s disease

IND

investigational new drug

MSC(s)

mesenchymal stem or marrow stromal cell(s)

MSC/BDNF

MSCs engineered to produce brain-derived neurotrophic factor

MSC/VEGF

MSCs engineered to secrete vascular endothelial growth factor