The therapeutic potential of ΦC31 integrase as a gene therapy system

INTRODUCTION: The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells. AREAS COVERED: The φC31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells. We focus on work with the φC31 integrase system during the past 12-18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells. EXPERT OPINION: We conclude that the φC31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The φC31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.     

Nanoscaffold based stem cell regeneration therapy: recent advancement and future potential

Importance of the field: Over the past years, extensive research has been directed towards tissue engineering using conventional scaffolds. In-depth studies in this field have led to the realization that in vivo cells interact with the extracellular matrix, composed of nanofibers at sub-micron scale, which not only provides the mechanical support to the cells but also plays a key role in regulation of cellular behavior. This has led to the development of nanofibrous scaffold (NFS) technology which in combination with stem cells is emerging as an important tool in the development of tissue engineering and regenerative medicine.

Areas covered in this review: This review summarizes the three methods of nanofibrous scaffold preparation and provides a state-of-the-art update on the recent advancement in the use of nanoscaffolds in stem cell regeneration therapy.

What the reader will gain: The review gives the reader an insight on nanoscaffold based therapy methods, such as how these scaffolds can potentially be designed and used in successful development of stem cell based therapies.

Take home message: NFS technology when coupled with stem cells and exploited in the right way has a strong potential of being used in stem cell based regenerative medicine.     

Progress of co-culture systems in cartilage regeneration

ABSTRACT

Introduction: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering.

Areas covered: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms.

Expert opinion: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.     

New approaches to expand hematopoietic stem and progenitor cells

Introduction: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages, and are currently the foundation of HSC transplantation therapy. A variety of methods have recently been explored to find a way to expand hematopoietic stem and progenitor cells (HSCs/PCs) ex vivo in order to improve the efficiency and outcome of HSC transplantation.

Areas covered: Recent studies of HSCs/PCs have led to the development of new ways to detect and purify HSCs/PCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSCs. These findings have provided new approaches for expanding HSCs/PCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials.

Expert opinion: Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSCs/PCs and manipulation of such pathways by SMCs could establish efficient, cost-effective, riskless and robust methods for ex vivo expansion of HSCs/PCs. With these efforts, more sophisticated HSC transplantation would be possible in the near future.     

Human umbilical cord mesenchymal stem cells: an overview of their potential in cell-based therapy

Introduction: Human umbilical cord mesenchymal stem cells (HUC-MSCs) are one of the typical adult stem cells; they have superiorities including low immunogenicity, non-invasive harvest procedure, easy expansion in vitro, and ethical access compared with stem cells from other sources. Therefore, HUC-MSCs are a promising candidate for cell-based therapy.

Areas covered: Here we reviewed the development of stem cell-based therapy, the manufacturing and banking process of HUC-MSCs, the emerging clinical studies in the field of cancer, central nervous system diseases, liver diseases and graft-versus-host disease, the potential therapeutic mechanisms, as well as challenges of HUC-MSCs in clinical translation.

Expert opinion: HUC-MSCs seem to be an optimal choice for stem cell-based therapy. However, before the cells translate from basic to clinical research, some problems still remain to be solved: i) building regulatory guidelines as well as an efficient and safe manufacturing procedure; ii) establishing donor’s genetic testing and long-term closely monitoring system; iii) conducting further clinical trials to determine the optimum and standard dosage, time, route, frequency and many other technical issues of HUC-MSCs transplantation.     

Advances in improving fertility in women through stem cell-based clinical platforms

Introduction: Due to their regenerative ability, stem cells are looked at as a promising tool for improving infertility treatments in women. As the main limiting factor in female fertility is represented by the decrease of ovarian reserve, the main goals of stem cell-based clinical platforms would be to obtain in vitro or in vivo neo-oogenesis. Refractory endometrial factor infertility also represents an obstacle for female reproduction for which stem cells might provide novel treatment strategies.

Areas covered: A systematic search of the literature was performed on MEDLINE/PubMed database to identify relevant articles using stem-cell based clinical or research platforms in the field of female infertility.

Expert opinion: In vitro oogenesis has not so far developed beyond the stage of oocyte-like cells whose normal progression to mature oocytes and ability to be fertilized was not proved. Extensive epigenetic programming of gamete precursors and the complex interactions between somatic and germ cells required for human oogenesis likely represent the main obstacles in stem-cell-based neo-oogenesis. Also resuming oogenesis in vivo in adulthood still appears a distant hypothesis, as there is still a lack of consensus about the existence and functionality of adult ovarian stem cells.     

Update on gene and stem cell therapy approaches for spinal muscular atrophy

Introduction: Spinal muscular atrophy (SMA) is the leading genetic cause of pediatric death to which at present there is no effective therapeutic. The genetic defect is well characterized as a mutation in exon 7 of the survival of motor neuron (SMN) gene. The current gene therapy approach focuses on two main methodologies, the replacement of SMN1 or augmentation of SMN2 readthrough. The most promising of the current work focuses on the delivery of SMN via AAV9 vectors via intravenous delivery.

Areas covered: In the review the authors examine the current research in the field of stem cell and gene therapy approaches for SMA. Also focusing on delivery methods, timing of administration and general caveats that must be considered with translational work for SMA.

Expert opinion: Gene therapy currently offers the most promising avenue of research for a successful therapeutic for SMA. There are many important practical and ethical considerations which must be carefully considered when dealing with clinical trial in infants such as the invasiveness of the surgery, the correct patient cohort and the potential risks.     

Perivascular cells as mesenchymal stem cells

Importance of the field: Mesenchymal stem cells are multipotent adult stem cell populations that have broad differentiation plasticity and immunosuppressive potential that render them of great importance in cell-based therapies. They are identified by in vitro characteristics based on their differentiation potential for clinical approaches while their biological properties and in vivo identities are often less understood.

Areas covered in this review: Recent research carried out in the last decade on mesenchymal stem cell biology suggests that mesenchymal stem cells from various tissues reside in a perivascular location and these can be identified as pericytes that function as mural cells in microvessels.

What the reader will gain: This review covers recent progress on understanding the link between pericytes and mesenchymal stem cells discussing specific points such as response to injury and tissue-specific functions.

Take home message: Despite a long and controversial history, there is a growing acceptance that perivascular cells are connected with mesenchymal stem cells, all that is really lacking is genetic evidence to show differentiation of pericytes into different cells types.     

An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling

The specific targeting of cells to sites of tissue damage in vivo is a major challenge precluding the success of stem cell‐based therapies. Magnetic particle‐based targeting may provide a solution. Our aim was to provide a model system to study the trapping and potential targeting of human mesenchymal stem cells (MSCs) during in vitro fluid flow, which ultimately will inform cell targeting in vivo. In this system magnet arrays were used to trap superparamagnetic iron oxide particle‐doped MSCs. The in vitro experiments demonstrated successful cell trapping, where the volume of cells trapped increased with magnetic particle concentration and decreased with increasing flow rate. Analysis of gene expression revealed significant increases in COL1A2 and SOX9. Using principles established in vitro, a proof‐of‐concept in vivo experiment demonstrated that magnetic particle‐doped, luciferase‐expressing MSCs were trapped by an implanted magnet in a subcutaneous wound model in nude mice. Our results demonstrate the effectiveness of using an in vitro model for testing superparamagnetic iron oxide particles to develop successful MSC targeting strategies during fluid flow, which ultimately can be translated to in vivo targeted delivery of cells via the circulation in a variety of tissue‐repair models. Copyright © 2012 John Wiley & Sons, Ltd.     

Potential benefits of allogeneic bone marrow mesenchymal stem cells for wound healing

Introduction: It is becoming increasingly evident that select adult stem cells have the capacity to participate in repair and regeneration of damaged and/or diseased tissues. Mesenchymal stem cells have been among the most studied adult stem cells for the treatment of a variety of conditions, including wound healing.

Areas covered: Mesenchymal stem cell features potentially beneficial to cutaneous wound healing applications are reviewed.

Expert opinion: Given their potential for in vitro expansion and immune modulatory effects, both autologous and allogeneic mesenchymal stem cells appear to be well suited as wound healing therapies. Allogeneic mesenchymal stem cells derived from young healthy donors could have particular advantage over autologous sources where age and systemic disease can be significant factors.     

Tissue engineering and microRNAs: future perspectives in regenerative medicine

Introduction: Tissue engineering is a growing area of biomedical research, holding great promise for a broad range of potential applications in the field of regenerative medicine. In recent decades, multiple tissue engineering strategies have been adopted to mimic and improve specific biological functions of tissues and organs, including biomimetic materials, drug-releasing scaffolds, stem cells, and dynamic culture systems. MicroRNAs (miRNAs), noncoding small RNAs that negatively regulate the expression of downstream target mRNAs, are considered a novel class of molecular targets and therapeutics that may play an important role in tissue engineering.

Areas covered: Herein, we highlight the latest achievements in regenerative medicine, focusing on the role of miRNAs as key modulators of gene expression, stem cell self-renewal, proliferation and differentiation, and eventually in driving cell fate decisions. Finally, we will discuss the contribution of miRNAs in regulating the rearrangement of the tissue microenvironment and angiogenesis, and the range of strategies for miRNA delivery into target cells and tissues.

Expert opinion: Manipulation of miRNAs is an alternative approach and an attractive strategy for controlling several aspects of tissue engineering, although some issues concerning their in vivo effects and optimal delivery methods still remain uncovered.     

Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.     

Recombinant AAV-directed gene therapy for type I glycogen storage diseases

Introduction: Glycogen storage disease (GSD) type Ia and Ib are disorders of impaired glucose homeostasis affecting the liver and kidney. GSD-Ib also affects neutrophils. Current dietary therapies cannot prevent long-term complications. In animal studies, recombinant adeno-associated virus (rAAV) vector-mediated gene therapy can correct or minimize multiple aspects of the disorders, offering hope for human gene therapy.

Areas covered: A summary of recent progress in rAAV-mediated gene therapy for GSD-I; strategies to improve rAAV-mediated gene delivery, transduction efficiency and immune avoidance; and vector refinements that improve expression.

Expert opinion: rAAV-mediated gene delivery to the liver can restore glucose homeostasis in preclinical models of GSD-I, but some long-term complications of the liver and kidney remain. Gene therapy for GSD-Ib is less advanced than for GSD-Ia and only transient correction of myeloid dysfunction has been achieved. A question remains as to whether a single rAAV vector can meet the expression efficiency and tropism required to treat all aspects of GSD-I, or if a multi-pronged approach is needed. An understanding of the strengths and weaknesses of rAAV vectors in the context of strategies to achieve efficient transduction of the liver, kidney and hematopoietic stem cells is required for treating GSD-I.     

Gene transfer mediated by stem cell grafts to treat CNS injury

Introduction: Stem cell transplantation holds promise for promoting anatomical repair and functional recovery after traumatic or ischemic injuries to the CNS. Harnessing stem cells with therapeutic genes of interest is regarded as an attractive approach to augment therapeutic benefits of stem cell grafts.

Areas covered: The advantage of stem-cell-mediated gene transfer is the engraftibility of stem cells that can ensure a long-term and stable expression of therapeutic genes. In addition, stem-cell–gene interaction may synergistically amplify therapeutic benefits. Delivery of classical neurotrophic factor genes provided neuroprotective and pro-regenerative effects in various injury models. Some studies employed therapeutic genes targeting post-injury microenvironment to support endogenous repair. Recent trials of stem-cell-mediated transfer of nonclassical growth factors showed relatively novel biological effects. Combinatorial strategies seem to have the potential to improve therapeutic efficacy.

Expert opinion: Future development of induced pluripotent stem cells and novel scaffolding biomaterials will greatly expedite the advances in ex vivo gene therapy to treat CNS injury. Before moving to a clinical stage, rigorous preclinical evaluations are needed to identify an optimal gene or gene combination in different injury settings. Improving the safety of viral vectors will be a critical prerequisite for the clinical translation.     

Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data

Introduction: Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources.

Areas covered: In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases.

Expert opinion: Enhanced results have been found when combining bone marrow–derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue–derived stem cells and umbilical cord tissue–derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.     

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.     

Stem cell therapies for wound healing

Introduction: Aberrant wound healing is a significant healthcare problem, posing a substantial burden on patients, their families, and the healthcare system. Existing treatment options remain only moderately effective and often fail to promote the closure of non-healing wounds in susceptible populations, such as aging and diabetic patients. Stem cell therapy has emerged as a promising treatment modality, with the potential to restore tissue to its pre-injured state. Of particular interest are mesenchymal stromal cells, which have been shown to accelerate wound healing by modulating the immune response and promoting angiogenesis.

Areas covered: This review provides an overview of wound healing and current methods for the management of chronic wounds, as well as the current state and considerations for optimizing stem cell therapy. Considerations include stem cell types, tissue source, donor selection, cell heterogeneity, delivery methods, and genetic engineering.

Expert opinion: A growing body of evidence has shown that delivery of stem cells, particularly mesenchymal stromal cells, has the potential to effectively improve the rate and quality of wound healing. However, significant additional basic and clinical research must be performed to optimize cell therapy, such as further elucidation of the therapeutic mechanisms of stem cells and standardization of clinical trial guidelines.     

Prospect of ultrasound-mediated gene delivery in cardiovascular applications

ABSTRACT

Introduction: The field of regenerative medicine has evolved over the years, investigating gene and stem/progenitor cell therapies to help address the increasing burden of cardiovascular disease (CVD). While the lack of success of gene therapy in clinical trials has dampened enthusiasm, the search continues for a successful and translatable gene therapy strategy for CVD. Ultrasound-mediated gene delivery (UMGD) is a non-invasive technique for gene delivery that utilizes gene-bearing carrier microbubbles and high power ultrasound to facilitate transfection in vivo. Many pre-clinical studies have shown benefit in animal models of CVD, but this has yet to be translated to human applications.

Areas covered: In this review, the basic principles of UMGD will be examined along with an overview of pre-clinical studies to date in CVD, focusing on cardiac and vascular applications and key findings. In addition, the potential path to the clinical translation of UMGD is discussed.

Expert opinion: Ultrasound-mediated gene delivery holds promise as a non-invasive technique for gene delivery in CVD, with the ability to deliver multiple genes with repeated deliveries over time. If the substantial hurdles to clinical translation can be overcome, UMGD may prove to be a key aspect in the success of cardiovascular gene therapy in the future.     

Combinatorial RNA-based gene therapy for the treatment of HIV/AIDS

Introduction: HIV/AIDS continues to be a worldwide health problem and viral eradication has been an elusive goal. HIV+ patients are currently treated with combination antiretroviral therapy (cART) which is not curative. For many patients, cART is inaccessible, intolerable or unaffordable. Therefore, a new class of therapeutics for HIV is required to overcome these limitations. Cell and gene therapy for HIV has been proposed as a way to provide a functional cure for HIV in the form of a virus/infection resistant immune system.

Areas covered: In this review, the authors describe the standard therapy for HIV/AIDS, its limitations, current areas of investigation and the potential of hematopoietic stem cells modified with anti-HIV RNAs as a means to affect a functional cure for HIV.

Expert opinion: Cell and gene therapy for HIV/AIDS is a promising alternative to antiviral drug therapy and may provide a functional cure. In order to show clinical benefit, multiple mechanisms of inhibition of HIV entry and lifecycle are likely to be required. Among the most promising antiviral strategies is the use of transgenic RNA molecules that provide protection from HIV infection. When these molecules are delivered as gene-modified hematopoietic stem and progenitor cells, long-term repopulation of the patient's immune system with gene-modified progeny has been observed.