From bench to bedside: review of gene and cell-based therapies and the slow advancement into phase 3 clinical trials, with a focus on Aastrom's Ixmyelocel-T.

There is a large body of preclinical research demonstrating the efficacy of gene and cellular therapy for the potential treatment of severe (limb-threatening) peripheral arterial disease (PAD), including evidence for growth and transcription factors, monocytes, and mesenchymal stem cells. While preclinical research has advanced into early phase clinical trials in patients, few late-phase clinical trials have been conducted. The reasons for the slow progression of these therapies from bench to bedside are as complicated as the fields of gene and cellular therapies. The variety of tissue sources of stem cells (embryonic, adult bone marrow, umbilical cord, placenta, adipose tissue, etc.); autologous versus allogeneic donation; types of cells (hematopoietic, mesenchymal stromal, progenitor, and mixed populations); confusion and stigmatism by the public and patients regarding gene, protein, and stem cell therapy; scaling of manufacturing; and the changing regulatory environment all contribute to the small number of late phase (Phase 3) clinical trials and the lack of Food and Drug Administration (FDA) approvals. This review article provides an overview of the progression of research from gene therapy to the cellular therapy field as it applies to peripheral arterial disease, as well as the position of Aastrom's cellular therapy, ixmyelocel-T, within this field.

Introduction

The exploration of cellular therapies and their application to various diseases and conditions is not new. Bone marrow stem cells have been studied since the 1950s. Accepted treatments are primarily for hematopoietic stem cells (HSCs) collected from the bone marrow or the peripheral blood for transplantation and treatment of specific types of bone marrow cancers (leukemia, lymphoma, and myeloma). Since the 1950s and 60s, the trajectory of cellular research has climbed steadily upward but few cellular therapies are Food and Drug Administration (FDA) approved [1]. The reasons for the slow progression from bench to bedside are as complicated as the field of cellular therapy itself. The variety of tissue sources of stem cells (embryonic, adult bone marrow, umbilical cord, placenta, adipose tissue, etc.); autologous versus allogeneic donation; types of cells (hematopoietic, mesenchymal stromal, progenitor, and mixed populations); confusion and stigmatism by the public and patients regarding gene, protein, and stem cell therapy; scaling of manufacturing; and the changing regulatory environment all contribute to the small number of Phase 3 trials and lack of approvals.

This review article provides an overview of the progression of research from gene therapy to the cellular therapy field as it applies to peripheral arterial disease, as well as the position of Aastrom’s cellular therapy, ixmyelocel-T, within this field.

Historical Perspective of the Overlap Between Gene and Cell Therapies

Pioneering work by Folkman in the 1970s led to development and eventual approval of anti-angiogenic treatments for cancer [2]. The discovery of angiogenic growth factors and signaling molecules that initiate and promote growth and survival of new blood vessels [3] captured the attention of cardiovascular investigators, who began testing the effects of growth factor stimulation on perfusion and function of ischemic tissues, independent of macrovessel surgery. Within the same timeframe, pioneering work was accomplished by British [4] and American scientists [5] in the isolation and culture of pluripotent embryonic stem cells (ESCs) from the inner cell mass of pre-embryos. During the 1980s and 1990s, stem cell research exploded; however, controversy over human cloning and embryo destruction led to policies that restricted ESC research. Only three Phase 1 clinical trials using ESCs have been conducted since 2010 [6]; the 3 studies were for indications of macular dystrophy, macular degeneration, and spinal cord injury.

Clinical research conducted using stem cells and stem cell-derived therapy from sources considered to be non-controversial have included research of adult multipotent stem cells from bone marrow, adipose tissue, umbilical cord, placenta, and endometrial tissue. Diseases studied have included graft versus host, Crohn’s, osteogenesis imperfecta, Parkinson’s, Alzheimer’s, and diseases of the cardiovascular system such as myocardial infarction, heart failure, and peripheral arterial disease (PAD).

Pathophysiology of Severe Peripheral Arterial Disease

Severe PAD, or critical (limb-threatening) limb ischemia (CLI), occurs when arterial blood flow is restricted to such an extent that the nutritive requirements of tissue can’t be met [7]. Ordinarily, compensatory mechanisms, including capillary sprouting as well as arteriogenesis [8], alleviate the effects of the deprivation, but in patients with CLI these mechanisms are exhausted. Inadequate blood flow to the skin and surrounding tissues leads to endothelial dysfunction, chronic inflammation [7], and muscle damage [9, 10]. The net effect of these changes is the occurrence of rest pain, chronic non-healing wounds, and gangrene. Treatment of severe PAD usually involves an attempt to address restricted blood flow through lower extremity revascularization using open bypass surgery or endovascular percutaneous intervention. While patients have benefits like wound healing from restoration of blood flow, simply reinstating flow on a macrovascular level does not reverse the damage of the alteration of structure and function of the endothelium and surrounding tissues that has occurred with severe PAD [8]. Limitations of the surgical or endovascular approach includes the increased mortality risk with open bypass procedures, restenosis, re-occlusion, re-intervention, and continued pain and expense of extensive wound care [11-13]. In addition, approximately 40 % of patients are not eligible for revascularization [14], leaving an opening for filling an unmet medical need in these very ill patients. The 5-year mortality rate for the most severe form of PAD is 60 % [15], exceeding that of prostate cancer (<1 %) [16], breast cancer (11 %) [17], acute myocardial infarction (20 %) [18], colorectal cancer (36 %) [19], and stroke (41 %) [20]. Cellular therapies may provide a treatment solution that has the potential to address multiple aspects of severe PAD including reduction of inflammation, tissue remodeling, and increased perfusion.

The sections below provide a summary of the preclinical evidence and the results of clinical trials in severe PAD for each of the gene and cellular therapy types. Table 1 provides a summary of ongoing or completed Phase 3 clinical trials by gene or cellular therapy type, Phase 2 clinical trials ongoing or completed and reported in the literature, and unique ongoing pilot studies.

Table 1

Clinical trials by gene or cellular therapy type

Sponsor/cell type	Phase/NCTa/Status/Design	Primary Efficacy Outcomes/Scientific Publications
Sponsors with PHASE 3 Studies
Aastrom Biosciences	Phase 3/ Active recruiting	AFS at 12 months
	NCT01483898
Multicellular; autologous (Ixmyelocel-T)	594 patients
	Randomized, DB, PBO-controlled
	Phase 2/Completed	Time to first occurrence of treatment failure (TTF) was significantly longer for patients treated with ixmyelocel-T compared to control patients (p = 0.0032, logrank test). TTF was defined as the earliest trial day on which any of the following treatment failure events occurred: major amputation of the injected leg, all-cause mortality (death), doubling of total wound surface area from baseline, and de novo gangrene. The survival curves diverged early and the difference between groups was maintained throughout the 12-month follow-up period. The Cox PH analysis gave a treatment HR = 0.381, 95 % CI = (0.195, 0.744), conveying a significant reduction in the risk of treatment failure in the ixmyelocel-T treatment group of approximately 62 % (p = 0.0047). The individual components of the treatment failure composite endpoint all trended in the same direction, favoring ixmyelocel-T treatment, with the exception of all-cause mortality that was the same in both treatment groups [91]
	NCT00468000
	72 patients
	Randomized, DB, PBO-controlled
Harvest Technologies	Phase 3/ Active recruiting	AFS at 6 months
	NCT01245335
	210 patients
(Device)	Randomized, DB, PBO-controlled
BM-MNC; autologous
	Phase 2/Ongoing	Time to amputation was longer in the BMAC group than in the placebo group (p = 0.067). In patients with tissue loss, treatment with BMAC demonstrated a lower amputation rate than placebo (39.1 % vs 71.4 %; p = 0.1337). Wound healing was not reported. Change in Rutherford Class (patients who improved at least one numeric category) showed that in Rutherford 4 patients, 81.8 % of the BMAC patients improved while 42.9 % of control patients improved (p = 0.0874); there was only a small difference in improvement (~3 %) between treatment groups in Rutherford 5 patients [97]
	NCT00498069
	48 patients
	Randomized, DB, PBO-controlled
Sanofi-Aventis	Phase 3/Completed (TAMARIS)	TAMARIS provided no evidence that NV1FGF is effective in reduction of amputation or death in patients with CLI [37].
Growth factor (FGF)	NCT00566657
	526 patients
	Randomized, DB, PBO-controlled
	NV1FGF	Similar rates of ulcer healing occurred with NV1FGF (19.6 %) and PBO (14.3 %; P = 0.514). The use of NV1FGF reduced by 2-fold the risk of all amputations [HR 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015) in the MITT study population (18 patients were excluded from efficacy analyses; the robustness of findings in relation to the occurrence of amputation, death, and AFS was confirmed in the total randomized population; however data were not shown). There was no statistically significant trend to suggest that NV1FGF reduced risk of death (HR 0.460; P = 0.105) [36]
	Phase 2/Completed (TALISMAN 201)
	NCT00368797
	125 patients
	DB, randomized, PBO-controlled
Sponsors or Investigators with PHASE 2 Studies
Makinen, 2002	Phase 2/Completed	Positive vascularity; negative restenosis rate, Rutherford class, and ABI [32]
VEGF-165	54 patients
(adnovirus/plasmid)	DB, randomized, PBO-controlled
RAVE	Phase 2/Completed	Negative for peak walking time, ABI, claudication onset time, and Quality of life. Treated arm associated with dose-dependent peripheral edema [33]
Rajagopalan, 2003	105 patients
VEGF-121 (adenovirus)
	DB, randomized, PBO-controlled
GRONINGEN	Phase 2/Completed	Negative for amputation rates; improvement in ulcer healing and ABI [98]
Kusumanto, 2006
VEGF-165 (plasmid)
	54 diabetic patients
	DB, randomized, PBO-controlled
Viromed Co, Ltd	Phase 2/Active recruiting	Difference in pain level between baseline and 9 month follow-up as determined by VAS
	NCT01064440
Growth factor HGF (2 isoforms HGF 728 and HGF 723)	50 patients
	Randomized, DB, PBO-controlled
AnGes	Phase 2/Completed	Change in TBI significantly improved from baseline at 6 months in the HGF-treated group compared with placebo (0.05 ± 0.05 vs -0.17 ± 0.04; P = 0.047). Change in VAS from baseline at 6 months was also significantly improved in the HGF-treated group compared with placebo. Complete ulcer healing at 12 months occurred in 31 % of the HGF group and 0 % of placebo (P = 0.04). There was no difference in major amputation of the treated limb (HGF 29 % vs placebo 33 %) or mortality at 12 months (HGF 19 % vs placebo 17 %) [43].
HGF and modified HGF
	NCT00189540
	27 patients
	DB, Randomized, PBO-Controlled
	Phase 2/Completed (HGF-STAT)	TcPO2 (mean SE) increased at 6 months in the high-dose group (24.0_4.2 mm Hg, 95 % CI 15.5 to 32.4 mm Hg) compared with the placebo (9.4_4.2 mm Hg, 95 % CI 0.9 to 17.8), low-dose (11.1_3.7 mm Hg, CI 3.7 to 18.7 mm Hg), and middle-dose (7.3_4.8 mm Hg, CI _2.2 to 17.0 mm Hg) groups (ANCOVA P_0.0015). There was no difference between groups in secondary end points, including ankle brachial index, toe brachial index, pain relief, wound healing, or major amputation [44]
	NCT00060892
	104 patients
	DB, randomized, PBO-controlled
Genzyme	Ad2/HIF-1α/VP16	No significant differences in claudication onset time, ABI, or quality of life measurements between placebo and each of 4 HIF-1α dose groups [99]
WALK study	NCT00117650
Transcription factor (HIF-1)	Phase 2/Completed
	289 patients
	Intermittent claudication
	Randomized, DB, PBO-controlled
Juventas Therapeutics, Inc	SDF1	Tracking of major/minor amputations, overall survival, QoL, ulcer healing, and pressure assessments
	Phase 2/Active recruiting
Cytokine (SDF) in 1 trial and BM-MNC; autologous for 1 trial	NCT01410331
UMC Utrecht for BM-MNC study
	48 patients
	BM-MNC autologous	Major amputation (primary), number and extent of leg ulcers, resolution of rest pain, improvement of ABI, improvement TcPO2, QoL [100]
	Phase 1–2/Not enrolling
	NCT00371371
	DB, randomized, PBO-controlled
	109–160 patients
TACT study group	Pilot study and Phase 2a	At 4 weeks, ABI was significantly improved in legs injected with BM-MNC compared to PB-MNC. Similar improvements were seen for transcutaneous oxygen pressure and pain free walking time. These improvements were sustained at 24 weeks [101]
BM-MNC and PB-MNC	NCT00145262
	25 patients with unilateral disease in pilot study who were injected with BM-MNC, followed by 22 patients with bilateral disease who were randomly injected with BM-MNC in one leg and PB-MNC in the other leg as control.
Johann Wolfgang Goethe University Hospitals (Germany)	Phase 1–2/Completed	Intra-arterial administration of BM-MNC did not significantly increase ABI. Cell therapy was associated with significantly improved ulcer healing versus placebo, and reduced rest pain versus placebo within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups [102]
	NCT00282646
BM-MNC; autologous
	40 patients
	DB, randomized, PBO-controlled
Losordo, Douglas, M.D.	Phase 1–2/Completed	A single administration of unmodified, autologous CD34 cell therapy was associated with significantly reduced rates of amputation in subjects with Rutherford class 4 and 5 critical limb ischemia. Ongoing analysis will examine additional endpoints, and will determine sample size and suitability of this therapy for a phase III study in patients with critical limb ischemia [103]
Baxter Healthcare Corporation	NCT00616980
BM-MNC	28 patients
	Multicenter, DB, PBO-controlled
Biomet Biologics, LLC	Phase not stated/Active recruiting	AFS at 1 year
Device	NCT01049919
	152 patients
	Randomized, DB, PBO-controlled
BM-MNC	Phase 1–2/Completed	AFS at 1 year was 86.3 %. There was a significant increase in FTP and TBI, and a trend in improvement in ABI. The VascuQol questionnaire demonstrated significant improvement in quality of life, and 3 of 9 ulcers (33 %) healed completely [104]
	29 patients (30 limbs)
	OL, nonrandomized
Washington University School of Medicine	Investigator Sponsored Study	AFS at 1 year
	Phase 3/Ongoing; not recruiting
STEMPAD study	NCT00797056
G-CSF mobilized PB-MNC	60 patients
	Randomized, DB, PBO-controlled
Pluristem, Ltd	Phase 2/Not yet recruiting	Primary outcome: Log ratio of week 52 maximal walking distance(MWD) to baseline MWD
MSCs; allogeneic	NCT01679990
	150 patients
	DB, PBO-controlled
Pilot Studies
Medistem, Inc. (Device)	Phase 1–2/Not yet recruiting in US	Improvements post-treatment in rest pain (VAS), toe pressure and ABI, transcutaneous oximetry and ulcer status (with picture) at 12 weeks
Endometrial regenerative cells/allogeneic	NCT01558908
	15 patients
	Open-label; no comparator
	Initiated in China (2 patients treated in early July 2012)
Investigator Trial (Northwestern University)	Phase 1/Active recruiting	ABI (15 % will be considered improvement), healing of ischemic ulcers, and decreased pain-follow-up at 1, 6, 12, and 24 months
	NCT01019681
Cord Blood injection IM
	25 patients
	OL, nonrandomized

aNCT (National Clinical Trial Number) if available

ABI ankle brachial index, DB double-blind, PBO placebo

Gene Therapy

Angiogenic Growth and Transcription Factors

The biological process for wound repair is initiated immediately after injury by release of various growth factors and cytokines. Therefore, angiogenic growth factors have been likely candidates for biological therapy and have been studied for the treatment of ischemic disease. Isner et al. published the first preclinical studies of therapeutic angiogenesis in the treatment of limb ischemia in 1995 [21, 22]. Several growth and transcription factors have advanced from basic research into vascular clinical trials for the treatment of PAD. These include vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), hypoxia inducible factor-1α (HIF-1α), fibroblast growth factor (FGF), and stromal-derived factor (SDF-1). There is extensive preclinical research showing the angiogenic potential for each of these factors.

The VEGF family, comprised of seven major isoforms, is the most widely studied endothelial growth factor [23]. VEGF, a key promoter of angiogenesis, stimulates proliferation, migration, and vascular formation and mobilizes endothelial progenitor cells [24-28]. The therapeutic potential of VEGF has been shown in PAD models where bolus injection of VEGF increased blood perfusion and tissue oxygenation [29-31]. Clinical trials using various isoforms were primarily conducted during the early 2000s (Table 1). Only one of the placebo-controlled trials had a positive primary endpoint of vascularity as measured by digital subtraction angiography, but all secondary outcomes were negative relative to placebo [32]; all other trials were negative for the primary efficacy measures [23]. In addition, one of the clinical trials demonstrated dose-dependent peripheral edema in patients receiving VEGF [33].

FGF, consisting of 23 structurally related proteins, is a regulator of angiogenesis through potent mediation of endothelial cell migration, differentiation, and survival [34]. Ashara et al. was the first to show that FGF improved perfusion in an animal model of ischemia [35]. The results of 2 clinical trials have been published: a Phase 2 and a Phase 3 trial were completed using injections of NV1FGF, nonviral naked FGF plasmid DNA. Both studies were placebo-controlled. In the Phase 2 study there was a significant difference favoring NV1FGF treatment in the secondary endpoint of amputation-free survival (AFS); however, none of the other endpoints were supportive of this finding including wound healing, rest pain, or death [36]. The large Phase 3 study conducted with 525 patients was negative for all endpoints (Table 1) [37].

HGF is a cytokine known to regulate cell growth, motility, morphogenesis and angiogenesis through activation of tyrosine kinase [38, 39]. In preclinical models, HGF has been shown to induce robust collateral formation [40, 41]. Expression of HGF was found to be strongly upregulated in cells of the wound epidermis during healing of excisional wounds in rats [42]. Two placebo-controlled clinical studies were conducted evaluating treatment of CLI patients with HGF. In the first study of 27 patients, change in toe brachial index was significantly improved from baseline at 6 months of follow-up in the HGF-treated group compared with placebo, and there was complete ulcer healing at 12 months in 31 % of HGF-treated compared to 0 % of placebo patients [43]. In the second study conducted by the same investigational group, 106 CLI patients were randomized to placebo (N = 26) or to 1 of 4 doses of HGF (N = 78). In all evaluable groups, measurement of blood flow by transcutaneous oxygen tension (TcPO2) at 6 months increased from baseline in HGF-treated relative to placebo patients; the highest increase was in the high-dose HGF group. All other endpoints were negative [44]. A Phase 3 study is planned to begin in 2012 in 560 CLI patients with rest pain or tissue loss [45].

HIF-1α, a transcription factor, regulates oxygen homeostasis and metabolism through adaptive responses to hypoxia at the cellular level [46-51], coordinating effort on multiple pathways that regulate angiogenesis, including VEGF, as well as pathways relevant to cell survival and metabolism [52-55]. In preclinical studies using rabbit hindlimb ischemia, administration of HIF-1α increased collateral blood vessels, capillary density and regional blood flow [56], and in a murine diabetic model of CLI enhanced neovascularization, mobilized progenitors cells from the bone marrow, and improved tissue perfusion [57]. Only one Phase 2 HIF-1α trial in patients with PAD has published results at the time of this manuscript (see Table 1; NCT00117650); in this trial HIF-1α was not shown to be an effective treatment for patients with intermittent claudication, a less severe form of PAD than CLI.

SDF-1 is a chemokine that is rapidly overexpressed in response to tissue injury. In the ischemic mouse hind limb, SDF-1 was shown to enhance angiogenesis [58]. SDF-1 has unique properties compared with the effects of angiogenic growth factors, including the absence of significant mitogenic actions which may prevent uncontrolled endothelial cell proliferation and subsequent formation of enlarged tortuous vessels, common in VEGF-induced angiogenesis [59]. Rapid inactivation of the chemokine in the protease-rich environment of the ischemic limb was addressed in the design by Segers et al. of recombinant SDF-1 proteins carrying mutations that provide resistance to protease cleavage [60]. Only one clinical trial is currently being conducted exploring the therapy value of SDF-1 (Table 1). The study is a Phase 2 trial that is currently ongoing.

Cellular Therapies

Mononuclear Cell Fraction/Endothelial Progenitor Cells

The exploration of both autologous bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNCs) for the treatment of ischemic disease has been explored for more than 10 years. These mononuclear cells, also called endothelial progenitor cells (EPC), are obtained from the CD34+ stem cell fraction of adult bone marrow and peripheral blood through cell separation techniques, and have the same lineage as hematopoietic stem cells with several shared surface antigens including KDR, Tie-2/Tek, and CD34 [61]. Early evidence showed postnatal neovascularization activities in response to ischemia including response of resident endothelial cells, but also the proliferation, differentiation, migration, and incorporation of BM-derived EPCs [62-65]. It was shown in experiments that EPCs specifically home to sites of ischemia and incorporate into capillaries and interstitial arteries in models of limb and myocardial ischemia [63, 66, 67]. EPCs have been shown in preclinical studies to improve capillary density in hindlimb models of ischemia [61, 68]. A review of the clinical study literature by Sprengers in 2008 found 25 published reports of clinical studies of BM-MNCs or PB-MNCs [69]. Most of the studies were case or patient series, 1 was a pilot, and 2 were randomized clinical trials. Results from the 2 randomized trials are presented in Table 1. In a review of stem and progenitor cell therapy by Lawall [70], the author concluded that despite the limitations of published BM-MNC clinical studies (small number of patients, lack of control group, and differing primary efficacy measurements), the outcomes were remarkably consistent. Clinical course (wound healing, walking distance) and perfusion parameters (ankle brachial index [ABI], TcPO2) were consistent and positive across trials. There are 2 studies using BM-MNC therapy actively recruiting patients (Table 1).

Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) were first identified in bone marrow 40 years ago [71]. MSCs are plastic-adherent cells that were shown to differentiate into osteoblasts, adipocytes, and chondrocytes in the 1980s [72, 73]. Caplan et al. showed that surrounding conditions are critical for inducing MSC differentiation [73]. In the 1990s, Pittenger et al. demonstrated that individual adult human MSCs were capable of expanding to colonies while still retaining their multipotency [74]. An essential characteristic of MSCs is that they home to damaged tissues and have been shown to regulate immune and inflammatory responses at target sites [75-79]. Isolation of MSCs have been performed in tissues other than bone marrow, including peripheral blood [80], cord blood [81], adipose tissue [82], synovial membrane [83], and placenta tissue [84]. Baksh et al. showed that MSCs derived from different tissues show phenotype heterogeneity and different growth abilities but also show similarities, with the potential to differentiate into the classical mesenchymal lineages [85]. There are a broad number of indications under study for treatment with MSCs; at the time of this article there were 254 active clinical trials for MSCs listed on clinicaltrials.gov. Some of the studies use allogeneic sources of cells and some use autologous. Indications include graft versus host disease, heart failure, diabetes, Parkinson’s, arthritis, aplastic anemia, Crohn’s disease, and multiple sclerosis. Of the 254 studies, 13 are listed with an indication of CLI and 1 with an indication of intermittent claudication (IC). Almost all of these studies are being conducted outside of the United States (U.S.), primarily in India and China; only the IC study has sites listed for recruitment in the U.S. There are no published results in peer-reviewed journals for early clinical trials using MSC-only therapy for the treatment of PAD.

Mixed Cellular Therapy

One hypothesis is that a mixture of regenerative cell types like MSCs (CD90+) and alternatively activated or M2 macrophages (CD14+ that express CD206+ and/or CD163+), rather than a single cell type, may be required to promote long-term tissue regeneration and repair [86, 87]. Aastrom Biosciences, Inc. manufactures ixmyelocel-T, an autologous multicellular therapy expanded from a patient’s own bone marrow. Ixmyelocel-T is composed of a mixture of cell types that include those expected to be found in the BM-MNC population. These include myeloid cells (granulocytes, monocytes, and mixed myeloid progenitors) and lymphoid cells (T cells, B cells, and mixed lymphoid progenitors) that express CD45 on the cell surface, and CD90+ MSCs/stromal cells, and CD45+CD14+ autofluorescent+ (CD14+Auto+) macrophages. While the cell types are similar to those found in the BM-MNC population, the numbers of CD90+ and CD14+Auto+ cells are significantly greater in ixmyelocel-T due to expansion during the manufacturing process. The manufacturing process and cell characterization of the product have been described previously [88, 89]. In in vitro studies it has been demonstrated that ixmyelocel-T produces anti-inflammatory cytokines which may aid in the healing process [90]. A Phase 2b clinical study (RESTORE-CLI) was successfully completed in 2011 [91], with results presented at the American Heart Association Scientific Sessions 2011 [92]. RESTORE-CLI was not powered to show statistical significance for efficacy endpoints; despite that limitation; however, there was a statistically significant difference in the time to first occurrence of treatment failure. The treatment failure composite, which consisted of major amputation of the index leg, all-cause mortality, doubling of wound total surface area from baseline and de novo gangrene can be considered a Phase 2 surrogate for the Phase 3 AFS (major amputation of the index leg, all-cause mortality) endpoint since tissue loss and gangrene are associated with higher rates of amputation and lower rates of survival [93]. Time to first occurrence of treatment failure is the earliest day at which any of the treatment failure events occurred. There was a 62 % risk reduction in treatment failure over the 12-month follow-up in the ixmyelocel-T group compared to the control group (hazard ratio 0.38, 95 % confidence interval = 0.20 to 0.74). The individual components of the treatment failure composite endpoint all trended in the same direction, favoring ixmyelocel-T treatment, with the exception of all-cause mortality that was the same in both treatment groups. A pivotal Phase 3 clinical trial (REVIVE) is being conducted under a Special Protocol Assessment (SPA) approved by the FDA, and began screening patients in 2012.

Discussion

There is a large body of preclinical research demonstrating the efficacy of gene and cellular therapy in peripheral arterial disease including evidence for growth and transcription factors, monocytes, and mesenchymal stem cells. However, thus far, clinical investigations have remained trapped in earlier phase studies, with the exception of fibroblast growth factor which advanced to a large-scale Phase 3 clinical trial. The disappointing results of this trial as well as the mixed positive and negative results from early clinical trials in both gene and cellular therapy, the complexity of the stem cell field, and the changing regulatory landscape have contributed to both the perception and the reality of the slow progression of research into later phase clinical trials.

Among the complicating factors are differing composition and biologic activities within the field of candidate therapies in the gene and stem cell fields. Gene therapy, the delivery of a single gene to the ischemic tissue of interest, requires expression of resident cells at the right time and place for efficacy [94]. Promotion of a single gene expression may not address the complexity of the underlying disease. Cellular therapies with adult stem cells have either autologous or allogeneic sources of cells, as well as differences in cell types. Allogeneic sources generally involve a single cell type such as MSCs. The expansion of the cells in vitro is essential for cost effectiveness and ‘off the shelf’ use. However, in vitro expansion may decrease the efficacy of the cells, causing dose to be an issue as well as engraftment potential of the cells. Repeat or multiple dosing may elicit an immune response. The requirements for follow-up of side effects and hurdles of regulatory oversight are more extensive for therapies using allogeneic cell sources. Autologous cell therapy can harvest single or multiple cell types. Using the patient’s own cells has advantages for safety, the maintenance of potency, and the potential for long-term engraftment and clinical effects. Multiple cell types have the potential to deliver multiple mechanisms to address complex diseases. Autologous cell products have more complex manufacturing and logistical issues, but there is the advantage of strict quality control for manufactured products over point of care devices. Autologous cell therapy developed using bedside devices that concentrate a larger volume bone marrow aspirate for reinjection to ischemic tissue are not as strictly quality controlled, but as devices have lower regulatory hurdles compared with manufactured autologous cell therapies.

The traditional drug development pathway of pharmacokinetic and preclinical modeling does not always translate well for stem cell products. As a result, much of the traditional ‘preclinical’ work must be done and will continue to be done within the framework of clinical trials, while still demonstrating proof of concept with in vitro studies and applicable in vivo models. Therefore, it is vitally important to communicate and use standardized protocols for the evaluation of efficacy and safety in both preclinical and clinical evaluation of cellular products. Early clinical trials used measurements of blood flow as the primary efficacy measure (e.g., ABI or TcPO2); however, there is poor correlation between leg blood flow and functional disability in patients with PAD [95]. The influence of the requirement by FDA to use AFS as the primary efficacy measurement in later phase clinical trials for severe PAD is reflected in the primary efficacy measures listed on clinicaltrials.gov for device trials as well as trials being conducted outside the U.S.

The past 15 years of clinical trials in gene and cell therapy for PAD have provided important knowledge and insights in regenerative medicine for vascular disease. There has been critical movement towards standardized, quality controlled, good manufacturing processes and protocols for the isolation and reintroduction of cells [6]. There are currently only two Phase 3 trials currently recruiting patients for the treatment of severe PAD with sites in the U.S. [96], including Aastrom’s trial with ixmyelocel-T, a multicellular expanded product, and a Harvest Technologies device trial using bedside concentration of BM-MNCs. Both of these trials use autologous bone marrow as the source of cells. In addition, a Phase 3 study is being planned for the evaluation of HGF [45]. The completion of these studies will add to the base of knowledge and provide new pieces of the regenerative medicine puzzle. Studies using allogeneic sources, including placental, cord blood, and endometrial tissue, remain in early-phase development.