Pancreatic cancer: gene therapy approaches and gene delivery systems

Importance of the field: Due to the absence of early diagnosis, the highly invasive and metastatic features and the lack of effective therapeutic modalities, the prognosis of patients with pancreatic cancer is poor. Gene therapy is currently regarded as a potential and promising therapeutic modality for pancreatic cancer.

Areas covered in this review: This article summarizes an update of gene therapy approaches and reviews the latest progress in gene delivery systems that have been tested on pancreatic cancer.

What the reader will gain: The treatment effectiveness of gene combination therapy is better than that of the regulation of single-gene or single gene therapy approaches. Naked DNA is limited because of degradation by intracellular and extracellular nucleases. Virus vectors show high transfection efficiency but are limited due to immunogenicity, inflammatory response and potential carcinogenicity. Non-viral vectors, such as cationic polymers or inorganic nanoparticles, show an important feature that they can be easily modified, and the progress of materials science will provide more and better non-viral vectors, accordingly improving the efficiency and safety of gene therapy, which will make them the most promising vectors for pancreatic cancer.     

Gene therapy for lysosomal storage disorders

Introduction: Lysosomal storage disorders (LSDs) encompass more than 50 distinct diseases, caused by defects in various aspects of lysosomal function. Neurodegeneration and/or dysmyelination are the hallmark of roughly 70% of LSDs. Gene therapy represents a promising approach for the treatment of CNS manifestations in LSDs, as it has the potential to provide a permanent source of the deficient enzyme, either by direct injection of vectors or by transplantation of gene-corrected cells. In this latter approach, the biology of neural stem/progenitor cells and hematopoietic cells might be exploited.

Areas covered: Based on an extensive literature search up until March 2011, the author reviews and discusses the progress, the crucial aspects and the major challenges towards the development of novel gene therapy strategies aimed to target the CNS, with particular attention to direct intracerebral gene delivery and transplantation of neural stem/progenitor cells.

Expert opinion: The implementation of viral vector delivery systems with specific tropism, regulated transgene expression, low immunogenicity and low genotoxic risk and the improvement in isolation and manipulation of relevant cell types to be transplanted, are fundamental challenges to the field. Also, combinatorial strategies might be required to achieve full correction in LSDs with neurological involvement.     

AAV-directed muscular dystrophy gene therapy

Importance of the field: Muscle-directed gene therapy for genetic muscle diseases can be performed by the recombinant adeno-associated viral (rAAV) vector delivery system to achieve long-term therapeutic gene transfer in all affected muscles.

Areas covered in this review: Recent progress in rAAV-vector-mediated muscle-directed gene transfer and associated techniques for the treatment of muscular dystrophies (MD). The review covers literature from the past 2 – 3 years.

What the reader will gain: rAAV-directed muscular dystrophy gene therapy can be achieved by mini-dystrophin replacement and exon-skipping strategies. The additional strategies of enhancing muscle regeneration and reducing inflammation in the muscle micro-environment should be useful to optimize therapeutic efficacy. This review compares the merits and shortcomings of different administration methods, promoters and experimental animals that will guide the choice of the appropriate strategy for clinical trials.

Take home message: Restoration of muscle histopathology and function has been performed using rAAV systemic gene delivery. In addition, the combination of gene replacement and adjuvant therapies in the future may be beneficial with regard to improving muscle regeneration and decreasing myofiber necrosis. The challenges faced by large animal model studies and in human trials arise from gene transfer efficiency and immune response, which may be overcome by optimizing the rAAV vectors utilized and the administration methods.     

TNFα gene delivery therapy for solid tumors

Importance of the field: Multimodality therapy, including adjuvant and neoadjuvant chemotherapy and radiotherapy, is now the mainstay of treatment for the majority of non-hematologic cancers. Host toxicity can, however, be significant, which may contribute to local and/or systemic failures. Novel adjunctive treatments that can limit systemic exposure while synergizing with standard therapy hold promise in the fight against an increasing number of cancers.

Areas covered in this review: We discuss a TNFα gene delivery system used to generate high levels of intratumoral TNFα, while limiting systemic exposure. The delivery system utilizes a replication-deficient adenoviral vector. When injected intratumorally and activated by external beam radiation, infected cells synthesize and locally secrete large amounts of TNFα.

What the reader will gain: This review will provide the reader with a thorough understanding of the gene-based TNFα delivery system with special emphasis on product characteristics, mechanisms of action, clinical efficacy, safety and tolerability.

Take home message: The TNFα gene delivery system holds promise as an adjunctive agent for improved local control and increasing resectability rates for many solid tumors. The completion of several ongoing randomized trials will help to better define the role for TNFα gene delivery therapy in the treatment of solid tumors.     

Strategies for short hairpin RNA delivery in cancer gene therapy

RNA interference (RNAi) gene silencing can be achieved by delivering vectors that transcribe short hairpin RNA (shRNA), which stably express small interfering RNA in target cells. Therefore, shRNA is of potential therapeutic use for inhibiting cancer cells, in which aberrant expression of certain mRNA's causes problems. However, this technique has not yet been developed for cancer therapy. The major problem for clinical use is lack of effective methods of delivery. In this article, we review the current strategies for shRNA delivery for target validation and their therapeutic uses in cancer to help further the understanding of challenges confronting shRNA technology, such as different principles of RNAi technology, basic construction of shRNA-expressing vectors and delivery barriers, which exist for both local and systemic delivery stratiges. Even if there are data showing that shRNA can be used in mice, we are still a long way from its application in human cancer therapy, because serious problems remain, including biodistribution and clearance of nanoparticles following systemic delivery of shRNA-expressing vectors.     

Mesenchymal stem cells at the intersection of cell and gene therapy

Importance of the field : Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secrete bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases.

Areas covered in this review: Findings demonstrating the immunomodulation, homing and paracrine activities of MSCs followed by a summary of the current research utilizing MSCs as a vector for gene therapy, focusing on skeletal disorders, but also cardiovascular disease, ischemic damage and cancer.

What the reader will gain: MSCs are a possible therapy for many diseases, especially those related to the musculoskeletal system, as a standalone treatment, or in combination with factors that enhance the abilities of these cells to migrate, survive or promote healing through anti-inflammatory and immunomodulatory effects, differentiation, angiogenesis or delivery of cytolytic or anabolic agents.

Take home message: Genetically-modified MSCs are a promising area of research that would be improved by focusing on the biology of MSCs that could lead to identification of the natural and engrafting MSC-niche and a consensus on how to isolate and expand MSCs for therapeutic purposes.     

Semliki Forest virus vectors for gene therapy

Semliki Forest virus (SFV) vectors transduce a broad range of mammalian and non-mammalian cells, generating high levels of transient expression of heterologous proteins. Generally, they induce apoptosis in mammalian host cells, leading to rapid cell death. These features have made SFV attractive for various gene therapy applications. Recombinant particles, naked RNA and plasmid DNA containing SFV replicons, demonstrate a strong immune response against recombinantly expressed proteins, which has shown protection against tumour challenges. Intratumoural injection of SFV particles has resulted in tumour regression. SFV vectors have been used for production of retrovirus-like particles. Recently, encapsulation of SFV particles into liposomes has generated highly efficient targeting to tumours. Novel SFV vectors based on point mutations in the non-structural genes, and avirulent SFV strains, have further widened the application range.     

Leptin gene therapy in the fight against diabetes

Importance of the field: The incidence of diabetes is increasing worldwide, yet current treatments are not always effective for all patient or disease types.

Areas covered in this review: Here, we summarize the biologic and clinical roles of leptin in diabetes, and discuss candidate viral vectors that may be employed in the clinical use of central leptin gene therapy for diabetes.

What the reader will gain: We discuss how studies on leptin, a regulator of the insulin–glucose axis, have significantly advanced our understanding of the roles of energy homeostasis and insulin resistance in the pathogeneses of metabolic syndrome and diabetes. Recent studies have demonstrated the long-term therapeutic effects of central leptin gene therapy in obesity and diabetes via decreased insulin resistance and increased glucose metabolism. Many of these studies have employed viral vectors, which afford high in vivo gene transduction efficiencies compared with non-viral vectors.

Take home message: Adeno-associated viral vectors are particularly well suited for central leptin gene therapy owing to their low toxicity and ability to drive transgene expression for extended periods.     

Modification of cardiovascular ion channels by gene therapy

Delivery of genes to the heart and vasculature for therapeutic purposes is an exciting strategy that is approaching clinical reality. Abnormalities of expression or function of ion channels is central to many cardiovascular diseases and gene delivery to modify ion channels is an appealing alternative to traditional therapy with small-molecule drugs. Potential therapeutic targets include hypertrophy and heart failure, atrioventricular node modification in atrial fibrillation, ventricular tachycardia and hypertension. Numerous approaches for gene delivery are under development, including use of tissue-specific promoters in viral vectors. For other applications, such as development of biological pacemakers, cells can be transduced with pacemaker genes in vitro, and then the cells implanted within the heart. There are short-term hurdles to therapeutic gene delivery to modify cardiovascular ion channels, but in the intermediate and longer term, the outlook is promising.     

Methods to improve cardiac gene therapy expression

Gene therapy strategies are becoming a valuable approach for the treatment of heart failure. Some trials are ongoing and others are being organized. Vascular access in clinical experimentation is still the chosen modality of delivery, but many other approaches are in research and development. A successful gene therapy strategy involves not only the choice of the right vector and gene, but also the correct delivery strategy that allows for transduction of the highest percentage of cardiomyocytes, limited spilling of virus into other organs and the possibility to correlate the amount of injected virus to the rate of the expression within the cardiac tissue. The authors will first concentrate on clarifying what the barriers are that the virus has to overcome in order to reach the nuclei of the target organs and methodologies that have been tested to improve the range of expression.     

The progress of AAV-mediated gene therapy in neuromuscular disorders

Introduction: The well-defined genetic causes and monogenetic nature of many neuromuscular disorders, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), present gene therapy as a prominent therapeutic approach. The novel variants of adeno-associated virus (AAV) can achieve satisfactory transduction efficiency of exogenous genes through the central nervous system and body-wide in skeletal muscle.

Areas covered: In this review, we summarize the strategies of AAV gene therapy that are currently under preclinical and clinical evaluation for the treatment of degenerative neuromuscular disorders, with a focus on diseases such as DMD and SMA. In addition to gene replacement strategy, we provide an overview of other approaches such as AAV-mediated RNA therapy and gene editing in the treatment of muscular dystrophies.

Expert opinion: AAV gene therapy has achieved striking therapeutic efficacy in clinical trials in infants with SMA. Promising results have also come from the preclinical studies in small and large animal models of DMD and several clinical trials are now on the way. This strategy shows great potential as a therapy for various neuromuscular disorders. Further studies are still required to confirm its long-term safety and improve the efficacy.     

Genotoxicity of retroviral hematopoietic stem cell gene therapy

Introduction: Retroviral vectors have been developed for hematopoietic stem cell (HSC) gene therapy and have successfully cured X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy, and Wiskott-Aldrich syndrome. However, in HSC gene therapy clinical trials, genotoxicity mediated by integrated vector proviruses has led to clonal expansion, and in some cases frank leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity with the aim of developing safer vectors and safer gene therapy protocols. These genotoxicity studies are critical to advancing HSC gene therapy.

Areas covered: This review provides an introduction to the mechanisms of retroviral vector genotoxicity. It also covers advances over the last 20 years in designing safer gene therapy vectors, and in integration site analysis in clinical trials and large animal models. Mechanisms of retroviral-mediated genotoxicity, and the risk factors that contribute to clonal expansion and leukemia in HSC gene therapy are introduced.

Expert opinion: Continued research on virus–host interactions and next-generation vectors should further improve the safety of future HSC gene therapy vectors and protocols.     

Promise of gene therapy to treat sickle cell disease

ABSTRACT

Introduction: Sickle cell anemia (SCA) is a hereditary blood disease caused by a single-gene mutation that affects millions of individuals world-wide. In this review, we focus on techniques to treat SCA by ex vivo genetic manipulation of hematopoietic stem/progenitor cells (HSPC), emphasizing replacement gene therapy and gene editing.

Areas covered: Viral transduction of an anti-sickling β-like globin gene has been tested in pre-clinical and early-phase clinical studies, and shows promising preliminary results. Targeted editing of endogenous genes by site-directed nucleases has been developed more recently, and several approaches also are nearing clinical translation.

Expert commentary: The indications and timing of gene therapy for SCA in lieu of supportive care treatment and allogeneic hematopoietic cell transplantation are still undefined. In addition, ensuring access to the treatment where the disease is endemic will present important challenges that must be addressed. Nonetheless, gene therapy and gene editing techniques have transformative potential as a universal curative option in SCA.     

Progress in gene and cell therapies for the neuronal ceroid lipofuscinoses

Introduction: The neuronal ceroid lipofuscinoses (NCLs) are a subset of lysosomal storage diseases (LSDs) that cause myoclonic epilepsy, loss of cognitive and motor function, degeneration of the retina leading to blindness, and early death. Most are caused by loss-of-function mutations in either lysosomal proteins or transmembrane proteins. Current therapies are supportive in nature. NCLs involving lysosomal enzymes are amenable to therapies that provide an exogenous source of protein, as has been used for other LSDs. Those that involve transmembrane proteins, however, require new approaches.

Areas covered: This review will discuss potential gene and cell therapy approaches that have been, are, or may be in development for these disorders and those that have entered clinical trials.

Expert opinion: In animal models, gene therapy approaches have produced remarkable improvements in neurological function and lifespan. However, a complete cure has not been reached for any NCL, and a better understanding of the limits of the current crop of vectors is needed to more fully address these diseases. The prospects for gene therapy, particularly those that can be delivered systemically and treat both the brain and peripheral tissue, are high. The future is beginning to look bright for NCL patients and their families.     

Non-viral gene therapy for neurological diseases, with an emphasis on targeted gene delivery

Non-viral gene therapy systems are considered safer than viral delivery. This article reviews recent research describing novel, non-viral gene delivery to the central nervous system, with a special emphasis on receptor mediated gene delivery using antibodies (termed immunogenes) to specific receptors. By using targeting agents such as antibodies that can be retrogradely transported within neurons, non-viral gene therapies can deliver genes to specific neurons protected by the blood brain barrier. Components of effective non-viral gene therapy are described including DNA/RNA carriers, receptor-mediated endocytosis, endosomal escape and nuclear entry. In addition, stealth agents such as polyethylene glycol that can be used to improve in-vivo delivery are discussed. The value of immunogenes as therapeutic agents for fatal diseases such as Amyotrophic Lateral Sclerosis is significant but further in-vivo work to confirm efficacy is required before truly effective therapies can be achieved.     

Enhanced T cell receptor gene therapy for cancer

Importance of the field: Adoptive therapy with T cell receptor- (TCR-) redirected T cells has shown efficacy in mouse tumor models and first responses in cancer patients. One prerequisite to elicit effective anti-tumor reactivity is the transfer of high-avidity T cells. Their generation, however, faces several technical difficulties. Target antigens are often expressed at low levels and their recognition requires the use of high-affine receptors. Yet, mainly low-affine TCRs have been isolated from tumor-infiltrating lymphocytes. Furthermore, upon transfer into a T cell the introduced receptor has to compete with the endogenous TCR.

Areas covered in this review: This review discusses how the functional avidity of TCR-modified T cells can be enhanced by i) increasing the amount of introduced TCR heterodimers on the cell surface; and ii) generating receptors with high affinity. Risks of TCR gene therapy and possible safety mechanisms are discussed.

What the reader will gain: The reader will gain an overview of the technical developments in TCR and T cell engineering.

Take home message: Despite technical obstacles, many advances have been made in the generation of high-avidity T cells expressing enhanced TCRs. Mouse studies and clinical trials will evaluate the effect of these improvements.     

Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation

Introduction: Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer.

Areas covered: A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer.

Expert opinion: Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.     

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.     

Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.