Current Gene Therapy - Volume 9, Issue 5, 2009

Gene therapy, defined as the use of genetic elements to treat diseases, is increasingly being used, with more than 1,300 clinical trials approved during the last decade [1]. These trials involve direct gene transfer into the target affected tissues, or the use of in vitro genetically engineered and expanded bone marrow stem cells. Deadly diseases such as adenosine deaminase deficiency (ADA) [2, 3], chronic granulomatous disease [4], and X-linked severe combined immunodeficiency (X-SCID) [5-7] have been successfully treated using autologous bone marrow cells transduced with retroviral vectors encoding the mutated genes. In spite of the biochemical, immunological, and clinical improvements in patient's lives, the development of leukemia in 5 out of 20 treated patients in the SCID-X1 gene therapy trials, involving the use of CD34+ autologous bone marrow cells transduced with retrovirus encoding the common γ-chain of cytokine receptors, called into question the overall safety of this therapeutic approach [8, 9]. It was later demonstrated that the development of leukemia in these patients was due to the insertion of the therapeutic gene in the vicinity of the LMO2 oncogene, and the unopposed growth advantage of transduced cells [10]. Importantly, out of the 5 patients who developed leukemia, one died in spite of treatment for leukemia, and 4 were in remission following chemotherapy, with a reconstituted immune system as a result of gene therapy. Thus, out of 20 patients who would have otherwise been restricted to life in a sterile bubble, 19 are able to have a normal life or a 95% success rate in an otherwise incurable disease. Since in the overall majority of the treated patients, the leukemia was successfully treated with chemotherapy, this highlights the tremendous success of gene therapy in treating a deadly disease, albeit at the expense of putative serious but treatable side effects. The events described above highlight the critical importance of very carefully assessing the possible risk vs. benefit ratios before attempting the clinical implementation of a novel therapeutic strategy, and the importance of learning lessons from clinical trials that will surely lead to improved genetic therapies. Another recently very highly publicized trial involved a Phase I-II gene therapy trial for rheumatoid arthritis [11] utilizing tgAAC94, adeno-associated vector (AAV), in which one of the patients enrolled in the trial died. Although the death was initially suspected to be potentially related to gene therapy, detailed investigations concluded that the most likely cause of death was disseminated, recrudescent fungal infection with Histoplasma capsulatum [12]. As these investigations regarding the details surrounding this patient's death unraveled, a number of questions were posed, i.e., should this patient have been enrolled in this trial given the substantial immunosuppressive treatment this patient was receiving to control her disease, were the inclusion-exclusion criteria sufficiently stringent, was the risk vs. benefit ratio properly evaluated, were the channels of communication between all parties involved appropriate? Curiously however, the fact that the gene therapy was being developed to selectively treat the affected joints, without having to expose patients to the very high incidence of serious adverse events caused by the systemic immune suppressants needed, was mostly missed by all reports.

Helper-dependent adenoviral vectors (HDAd) have several characteristics making them very attractive for human gene therapy. These vectors are completely devoid of viral coding sequences and are able to mediate high efficiency transduction in vivo to direct high level transgene expression with negligible chronic toxicity. Progress towards liver and lung directed gene therapy with HDAd as well as the current obstacles facing human applications and possible strategies to overcome these obstacles are discussed.

Oncolytic viral therapy is a promising biological therapy for the treatment of cancer. Recent advances in genetic engineering have facilitated the construction of custom-built oncolytic viruses that can be exquisitely targeted to tumors by exploiting each cancer's unique biology and their efficacy can be further enhanced by “arming” them with additional therapeutic genes. Such an approach allows the virus to unload its “therapeutic cargo” at the tumor site, thereby enhancing its anti-neoplastic properties. While several clever strategies have been recently described using genes that can induce cellular apoptosis/suicide and/or facilitate tumor/virus imaging, viruses armed with genes that also affect the tumor microenvironment present an exciting and promising approach to therapy. In this review we discuss recently developed oncolytic viruses armed with genes encoding for angiostatic factors, inflammatory cytokines, or proteases that modulate the extracellular matrix to regulate tumor vascularization, anti-tumor immune responses and viral spread throughout the solid tumor.

With the introduction of sophisticated tools of molecular biology, prodrug activating gene therapies have evolved as a novel therapeutic option for high-grade malignant gliomas. Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) is an extensively studied form of cytotoxic gene therapies. It is especially applicable for localized cancers, such as malignant glioma because of its restricted anatomical location and absence of metastasis. The early successes in the treatment of experimental malignant gliomas in the 1990s, gave impetus to further test this approach in this devastating disease. In malignant glioma, the recurrence after conventional therapy is inevitable, due to the residual cells in the tumor bed. The fascinating feature of adenoviral HSV-tk is that it attacks the residual dividing tumor cells without affecting the non-dividing neurons and furthermore, exploits them to destroy the malignant cells via so-called bystander- effect. Clinical Phase I and II studies have shown significant survival advantage and excellent safety profile when compared to conventional treatments. Thus, the adenoviral mediated HSV-tk gene therapy is a promising new adjuvant treatment for patients with operable high-grade glioma.

A large majority of Phase III, large scale, clinical trials will fail, including gene therapy trials. This paper attempts to address some of the causes that may have inadvertently led to such a high failure rate. After briefly reviewing the detailed and high quality work that goes both into the preparation and conduct of such large Phase III clinical trials, and the preclinical science that is used to support and originate such trials, this paper proposes a novel approach to translational medicine which would increase the predictability of success of Phase III clinical trials. We propose that a likely cause of such failures is the lack of “robustness” in the preclinical science underpinning the Phase I/II and III clinical trials. Robustness is defined as stability/reproducibility in the face of challenges. Many times preclinical experiments are tested in a very narrow set of experimental conditions. Thus, when such approaches are finally tested in the context of human disease, the challenge provided by the varied age of patients, the complex genetic makeup of human populations, and the complexities of the diseases to be treated provide challenges which were never tested or modeled. We believe that the introduction of revised approaches to preclinical science, including the use of the latest developments in statistical, scientific, mathematical, and biological models, ought to lead to more robust preclinical experimentation with its subsequent translation, to more robust Phase III clinical trials.

Glial cell line-derived neurotrophic factor (GDNF) family of ligands (GDFLs) as well as other trophic factors have, in animal models of Parkinson's disease (PD), demonstrated the potential for excellent ameliorative properties. Clinical trials that have mechanically injected GDNF intracerebrally, while demonstrating relative safety, have been clinically disappointing to date. Likewise, recombinant adeno-associated virus (rAAV) delivered neurturin (cere-120) has also been demonstrated to be safe in humans, however clinical results have been negative. The failure of the major clinical trials has cast some doubt in the field about trophic factor delivery for the treatment of PD. In this review, we make the case that GDFLs are likely to function only when there are remaining dopamine neurons in the nigrostriatal pathway as opposed to other candidate modes of action. Thus, it is our view that utilizing earlier stage PD patients who have significant nigrostriatal dopamine innervation remaining would be more ideal to demonstrate the efficacy of GDFLs. This is particularly true when considering a novel delivery method such as gene transfer. However, if earlier stage patients are to be enrolled in GDFL gene transfer trials, then a much better safety profile must be demonstrated by preclinical experiments. One important safety advance might be the use of an external regulation system to control the expression level of the transgene. However, gene regulation systems pose unique safety issues and we will discuss these in detail. It is our view that GDFLs still remain as a promising therapeutic approach for PD.

Glioblastoma multiforme is the most common primary intracranial tumor in humans. Despite continued advances in cancer therapy, the outcome for patients diagnosed with this disease remains bleak. Novel treatments involving the use of conditionally replicating adenoviruses (CRAds) to target malignant brain tumors have undergone extensive research and proven to be a promising mode of glioblastoma therapy. CRAds are genetically manipulated to replicate within tumor cells, exhibiting a high degree of infectivity, cytotoxicity, and transgene expression. While the use of various CRAds has been deemed safe for intracranial injection in preclinical trials, a significant therapeutic effect has yet to be seen in patients. This shortcoming stems from the distribution limitations involved with local delivery of virolytic agents. To enhance this modality of treatment, stem cells have been explored as cellular vehicles in virotherapy applications, given that they possess an intrinsic tropism for malignant brain tumors. Stem cell loaded CRAd delivery offers a more specific and effective method of targeting disseminated tumor cells and forms the basis for this review.

Adoptive T cell therapies can produce objective clinical responses in patients with hematologic and solid malignancies. Genetic manipulation of T lymphocytes has been proposed as a means of increasing the potency and range of this anti-tumor activity. We now review how coupling expression of transgenic receptors with countermeasures against potent tumor immune evasion strategies is proving highly effective in pre-clinical models and describe how these approaches are being evaluated in human subjects.

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults. Despite significant advances in treatment and intensive research, the prognosis for patients with GBM remains poor. Therapeutic challenges for GBM include its invasive nature, the proximity of the tumor to vital brain structures often preventing total resection, and the resistance of recurrent GBM to conventional radiotherapy and chemotherapy. Gene therapy has been proposed as a useful adjuvant for GBM, to be used in conjunction with current treatment. Work from our laboratory has shown that combination of conditional cytotoxic with immunotherapeutic approaches for the treatment of GBM elicits regression of large intracranial tumor masses and anti-tumor immunological memory in syngeneic rodent models of GBM. In this review we examined the currently available animal models for GBM, including rodent transplantable models, endogenous rodent tumor models and spontaneous GBM in dogs. We discuss non-invasive surrogate end points to assess tumor progression and therapeutic efficacy, such as behavioral tests and circulating biomarkers. Growing preclinical and clinical data contradict the old dogma that cytotoxic anti-cancer therapy would lead to an immune-suppression that would impair the ability of the immune system to mount an anti-tumor response. The implications of the findings reviewed indicate that combination of cytotoxic therapy with immunotherapy will lead to synergistic antitumor efficacy with reduced neurotoxicity and supports the clinical implementation of combined cytotoxic-immunotherapeutic strategies for the treatment of patients with GBM.

Oncolytic adenoviruses are emerging as a promising alternative therapy for glioma patients and are currently being tested in clinic. In this review, we summarize our experience with gene-based therapy targeting RB pathway in gliomas. Our study has evolved from the development of RB-expressing adenoviral vectors to the characterization of the oncolytic effects on gliomas of the replication competent adenoviruses Delta-24, Delta-24-RGD and ICOVIR. We also review the successful combination of the viruses with chemotherapies that are routinely used in glioma patients, the efficacy of Delta-24-RGD against brain tumor stem cells, the newly described adenovirus-induced autophagy and the potential for the systemic delivery of the oncolytic viruses with human mesenchymal stem cells. Finally, we comment on the preclinical and clinical studies of p53 expressing adenoviral vector and the lessons learned from the experience of Onyx- 015, the first oncolytic adenovirus tested in clinical setting.

Therapeutic delivery to the central nervous system has challenged scientists and clinicians due to the difficulty in delivering molecules and genes in an efficient manner across the blood brain barrier (BBB). This has particularly hampered efforts to deliver therapeutics to widely dispersed neurons that perish in diseases such as Amyotrophic Lateral Sclerosis (ALS), a disease affecting motor neurons throughout the brainstem and the entire spinal cord. Gene therapy has offered several potential routes to overcome the difficulties in delivering therapeutics to the brain and spinal cord. Adenoassociated viral vectors (AAV) have taken center stage for gene delivery to the central nervous system, given their ability to express genes in post mitotic cells for long periods with minimal to no toxicity. This review will focus on recent approaches to treat motor neuron disease, in particular ALS using AAV vectors.