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.     

Gene therapy, bioengineered clotting factors and novel technologies for hemophilia treatment

The World Federation of Hemophilia estimates that of the 400,000 individuals worldwide with hemophilia, 300,000 receive either no, or very sporadic, treatment. Thus, considerable innovation will be required to provide cost-effective therapies/cures for all affected individuals. The high cost of prophylactic regimens hampers their widespread use, which further justifies the search for novel cost-effective therapies and ultimately a cure. Five gene transfer phase I clinical trials have been conducted using either direct in vivo gene delivery with viral vectors or ex vivo plasmid transfections and reimplantation of gene-engineered cells. Although there was evidence of gene transfer and therapeutic effects in some of these trials, stable expression of therapeutic factor VIII or FIX levels has not yet been obtained. Further improvements of the vectors and a better understanding of the immune consequences of gene transfer is warranted, as new trials are being initiated. Bioengineered clotting factors with increased stability and/or activity are being validated further in preclinical studies. Novel clotting factor formulations based on PEGylated liposomes with prolonged activities are being tested in the clinic, and are yielding encouraging results.     

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation

This gene transfer experiment is the first Parkinson's Disease (PD) protocol to be submitted to the Recombinant DNA Advisory Committee. The principal investigators have uniquely focused their careers on both pre-clinical work on gene transfer in the brain and clinical expertise in management and surgical treatment of patients with PD. They have extensively used rodent models of PD for proof-of-principle experiments on the utility of different vector systems. PD is an excellent target for gene therapy, because it is a complex acquired disease of unknown etiology (apart from some rare familial cases) yet it is characterized by a specific neuroanatomical pathology, the degeneration of dopamine neurons of the substantia nigra (SN) with loss of dopamine input to the striatum. This pathology results in focal changes in the function of several deep brain nuclei, which have been well-characterized in humans and animal models and which account for many of the motor symptoms of PD. Our original approaches, largely to validate in vivo gene transfer in the brain, were designed to facilitate dopamine transmission in the striatum using an AAV vector expressing dopamine-synthetic enzymes. Although these confirmed the safety and potential efficacy of AAV, complex patient responses to dopamine augmenting medication as well as poor results and complications of human transplant studies suggested that this would be a difficult and potentially dangerous clinical strategy using current approaches. Subsequently, we and others investigated the use of growth factors, including GDNF. These showed some encouraging effects on dopamine neuron survival and regeneration in both rodent and primate models; however, uncertain consequences of long-term growth factor expression and question regarding timing of therapy in the disease course must be resolved before any clinical study can be contemplated. We now propose to infuse into the subthalamic nucleus (STN) recombinant AAV vectors expressing the two isoforms of the enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), which synthesizes the major inhibitory neurotransmitter in the brain, GABA. The STN is a very small nucleus (140 cubic mm or 0.02% of the total brain volume, consisting of approximately 300,000 neurons) which is disinhibited in PD, leading to pathological excitation of its targets, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr). Increased GPi/SNpr outflow is believed responsible for many of the cardinal symptoms of PD, i.e., tremor, rigidity, bradykinesia, and gait disturbance. A large amount of data based on lesioning, electrical stimulation, and local drug infusion studies with GABA-agonists in human PD patients have reinforced this circuit model of PD and the central role of the STN. Moreover, the closest conventional surgical intervention to our proposal, deep brain stimulation (DBS) of the STN, has shown remarkable efficacy in even late stage PD, unlike the early failures associated with recombinant GDNF infusion or cell transplantation approaches in PD. We believe that our gene transfer strategy will not only palliate symptoms by inhibiting STN activity, as with DBS, but we also have evidence that the vector converts excitatory STN projections to inhibitory projections. This additional dampening of outflow GPi/SNpr outflow may provide an additional advantage over DBS. Moreover, of perhaps the greatest interest, our preclinical data suggests that this strategy may also be neuroprotective, so this therapy may slow the degeneration of dopaminergic neurons. We will use both GAD isoforms since both are typically expressed in inhibitory neurons in the brain, and our data suggest that the combination of both isoforms is likely to be most beneficial. Our preclinical data includes three model systems: (1) old, chronically lesioned parkinsonian rats in which intraSTN GAD gene transfer results not only in improvement in both drug-induced asymmetrical behavior (apomorphine symmetrical rotations), but also in spontaneous behaviors. In our second model, GAD gene transfer precedes the generation of a dopamine lesion. Here GAD gene transfer showed remarkable neuroprotection. Finally, we carried out a study where GAD-65 and GAD-67 were used separately in monkeys that were resistant to MPTP lesioning and hence showed minimal symptomatology. Nevertheless GAD gene transfer showed no adverse effects and small improvements in both Parkinson rating scales and activity measures were obtained. In the proposed clinical trial, all patients will have met criteria for and will have given consent for STN DBS elective surgery. Twenty patients will all receive DBS electrodes, but in addition they will be randomized into two groups, to receive either a solution containing rAAV-GAD, or a solution which consists just of the vector vehicle, physiological saline. Patients, care providers, and physicians will be blind as to which solution any one patient receives. All patients, regardless of group, will agree to not have the DBS activated until the completion and unblinding of the study. Patients will be assessed with a core clinical assessment program modeled on the CAPSIT, and in addition will also undergo a preop and several postop PET scans. At the conclusion of the study, if any patient with sufficient symptomatic improvement will be offered DBS removal if they so desire. Any patients with no benefit will simply have their stimulators activated, which would normally be appropriate therapy for them and which requires no additional operations. If any unforeseen symptoms occur from STN production of GABA, this might be controlled by blocking STN GABA release with DBS, or STN lesioning could be performed using the DBS electrode. Again, this treatment would not subject the patient to additional invasive brain surgery. The trial described here reflects an evolution in our thinking about the best strategy to make a positive impact in Parkinson Disease by minimizing risk and maximizing potential benefit. To our knowledge, this proposal represents the first truly blinded, completely controlled gene or cell therapy study in the brain, which still provides the patient with the same surgical procedure which they would normally receive and should not subject the patient to additional surgical procedures regardless of the success or failure of the study. This study first and foremost aims to maximally serve the safety interests of the individual patient while simultaneously serving the public interest in rigorously determining in a scientific fashion if gene therapy can be effective to any degree in treating Parkinson's disease.     

The single breath transfer factor (Tl,co) and the transfer coefficient (Kco): a window onto the pulmonary microcirculation

The transfer factor, Tl,co (with the transfer coefficient, Kco, also known as the transfer factor per unit alveolar volume, [Tl/Va]), is one of the most useful clinical tests of pulmonary function, the only one which specifically focuses on pulmonary microcirculation. It was originally devised in 1909 as a physiological tool to assess the diffusive capacity of the lung as a gas exchanger. It was subsequently developed as a clinical tool, but cumbersome analytical techniques delayed its introduction into clinical medicine until 1950s. The physiology of the carbon monoxide transfer factor (also called the diffusing capacity Dl,co) is based on the Roughton-Forster equation which partitions Dl,co, a conductance, into membrane (Dm) and red cell (thetaVc) diffusion conductances. Recent work (1987-2001) suggests that 70-80% of the resistance to CO (and O2) diffusion may reside in the red cell fraction. The clinical implication is that Tl,co and Kco are 'windows' onto the pulmonary microcirculation. As regards reference values for clinical use, Tl,co depends on age, height and gender. Kco, which is actually a rate constant, is independent of gender, and is affected principally by age. A schema is presented for the clinical interpretation of Tl,co. As Tl,co is derived from the product of Kco and the accessible alveolar volume (Va), examination of these two components (Kco and Va) will usually suggest a specific pathophysiological mechanism as the explanation for a reduction in Tl,co.     

The challenge of follow-up for clinical trials of somatic gene therapy.

Although extensive efforts have been made to optimize the safety of vectors for somatic gene transfer and somatic gene therapy, the safety of these agents must ultimately be assessed in clinical trials. A statistically significant assessment of safety will be complicated by the relatively small number of patients who will be enrolled in initial clinical trials, the need for long-term longitudinal follow-up of patients and perhaps their progeny, and the traditionally poor participation in long-term follow-up by many patients. This article reviews the potential risks of retroviral-mediated gene transfer, the statistical power required to assess the true incidence of potential complications, the number of patients who may participate in clinical trials involving retroviral vectors, and the factors that make thorough follow-up and uniform data ascertainment difficult. The role of the FDA in assessing the safety of retroviral vectors and the potential role of registries for patient tracking and data ascertainment are discussed.     

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.     

Gene therapy of monogenic and cardiovascular disorders

The concept of gene therapy involves the introduction of genetic material into patient cells to cure or alleviate the symptoms of a disease by complementing a damaged gene or by giving the cell a new function. The belief that gene therapy would soon reach the clinic has been widely spread, frequently resulting in controversies when these expectations were not met. Nevertheless, over the last 10-year period, the experience from a number of clinical trials has taught us that gene transfer is technically feasible, but that the gene delivery vehicles, or vectors, for the transfer of genetic material are still suboptimal and that treatment may have severe side effects. This review will provide examples of different genetic disorders for which gene therapy is an option and has been attempted. It will also briefly discuss the existing vector systems and mention their advantages and drawbacks.     

Gene therapy of monogenic and cardiovascular disorders

The concept of gene therapy involves the introduction of genetic material into patient cells to cure or alleviate the symptoms of a disease by complementing a damaged gene or by giving the cell a new function. The belief that gene therapy would soon reach the clinic has been widely spread, frequently resulting in controversies when these expectations were not met. Nevertheless, over the last 10-year period, the experience from a number of clinical trials has taught us that gene transfer is technically feasible, but that the gene delivery vehicles, or vectors, for the transfer of genetic material are still suboptimal and that treatment may have severe side effects. This review will provide examples of different genetic disorders for which gene therapy is an option and has been attempted. It will also briefly discuss the existing vector systems and mention their advantages and drawbacks.     

Retrovirus packaging cells

Retroviral vectors promote the efficient transfer of genes into a variety of cell types from many animal species. An important contribution to their utility was the development of retrovirus packaging cells, which allow the production of retroviral vectors in the absence of replication-competent virus. Because of their ability to transfer genes efficiently into cells that are difficult to transfect by other methods, retroviral vectors are prime candidates for gene transfer into human somatic cells. Indeed, a retroviral vector recently has been used to mark tumor infiltrating lymphocytes in patients with melanoma to follow the persistence and distribution of these cells following infusion into patients. Hopefully these vectors will soon be used for the treatment of disease by transfer of functional genes, or gene therapy. Here I will review the available packaging cell lines and their properties with a focus on their ultimate application to human gene therapy.     

Analysis of data from multiclinic trials

Because the clinics in a multiclinic randomized clinical trial represent neither fixed stratification effects nor random classificatory effects, the appropriate analysis of data from such a trial has been the subject of controversy and debate. The following are some of the elements of controversy that are discussed and for which some bases for resolution are proposed. Is it ever valid to ignore the effects of clinics in the analysis? Is it ever valid to drop clinics from the analysis? Is a multiclinic clinical trial similar in structure or not to a single-clinic clinical trial in which patients have been stratified on a classificatory factor? Assuming that clinics will be taken account of in the analysis, should it be the weighted or the unweighted average of within-clinic treatment differences that is to be taken as the best estimate of the overall difference between the treatments? How should the data be analyzed if there is evidence of treatment-by-clinic interaction?     

Therapeutic angiogenesis for myocardial ischemia

Therapeutic angiogenesis offers promise as a novel treatment for ischemic heart disease, particularly for patients who are not candidates for current methods of revascularization. The goal of treatment is both relief of symptoms of coronary artery disease and improvement of cardiac function by increasing perfusion to the ischemic region. Protein-based therapy with cytokines including vascular endothelial growth factor and fibroblast growth factor demonstrated functionally significant angiogenesis in several animal models. However, clinical trials have yielded largely disappointing results. The attenuated angiogenic response seen in clinical trials of patients with coronary artery disease may be due to multiple factors including endothelial dysfunction, particularly in the context of advanced atherosclerotic disease and associated comorbid conditions, regimens of single agents, as well as inefficiencies of current delivery methods. Gene therapy has several advantages over protein therapy and recent advances in gene transfer techniques have improved the feasibility of this approach. The safety and tolerability of therapeutic angiogenesis by gene transfer has been demonstrated in phase I clinical trials. The utility of therapeutic angiogenesis by gene transfer as a treatment option for ischemic cardiovascular disease will be determined by adequately powered, randomized, placebo-controlled Phase II and III clinical trials. Cell-based therapies offer yet another approach to therapeutic angiogenesis. Although it is a promising therapeutic strategy, additional preclinical studies are warranted to determine the optimal cell type to be administered, as well as the optimal delivery method. It is likely the optimal treatment will involve multiple agents as angiogenesis is a complex process involving a large cascade of cytokines, as well as cells and extracellular matrix, and administration of a single factor may be insufficient. The promise of therapeutic angiogenesis as a novel treatment for no-option patients should be approached with cautious optimism as the field progresses.     

T-cell receptor gene transfer by lentiviral vectors in adoptive cell therapy

Adoptive cell therapy can be envisioned as a promising strategy for tumour immunotherapy. However, existing protocols of adoptive cell therapy still require optimisation as many factors, such as specificity, avidity, level of differentiation and amount of transferred T lymphocytes, can influence their immunocompetence and in vivo functionality. In particular, the need to reduce the in vitro expansion phase and to obtain large numbers of tumour-reactive T cells, as a favourable condition for cancer regression, make TCR gene transfer a potentially ideal tool to overcome the limits of adoptive cell therapy strategies. Here, the authors review the state-of-the-art and recent advances in TCR transfer with particular emphasis on lentiviral vector systems. Initial data from preclinical models and recent clinical trials encourage optimisation of a safe, simplified and stable transfer system. In this regard, HIV-based vectors are emerging as good alternative candidates over the most widely used oncoretroviral vectors due to their peculiar molecular features that fit the ideal conditions for donor T cell in vitro manipulation.     

Seminiferous tubule cannulation (STC): a new,sensitive technique for detecting gene transfer in developing sperm

As gene therapy vectors, strategies, and disease targets continue to expand and diversify, the likelihood that developing germ cells will be exposed to gene transfer vectors increases. Insertion of exogenous genetic material into the germ line might have devastating effects on normal development which could be heritable. Accordingly, it is important that vectors be tested for their potential to insert genes into developing gametes. Such tests are most difficult in males, where differentiating sperm are sequestered behind the blood-testis barrier. In this communication we report the development of a new technique, which we call seminiferous tubule cannulation (STC). We demonstrate that STC allows delivery of high quantities of gene therapy vector directly to spermatogenic cells without significantly disturbing the cytoarchitecture of the seminiferous tubule. To demonstrate the effectiveness of this technique, three promoters driving lacZ gene expression in adenovirus vectors were tested for their ability to transduce cells within the seminiferous tubule. Results indicate that the cytomegalovirus promoter, but not the Rous sarcoma virus or elongation factor 1alpha promoters, is active within the seminiferous tubule. Further development of this technique promises to lead to a standardized test for male germ cell transduction by gene therapy vectors.     

Proper use of antidepressants in the acute and maintenance phase treatment of depression

In spite of their different pharmacological profiles, a number of meta-analyses have shown that all antidepressants are equally effective in the treatment of major depression. However, there is the discordance between efficacy data and clinical observation. So then, how should we use antidepressants properly in the clinical setting? Toward rational pharmacotherapy, the selection of antidepressant medications will be based on their pharmacological profiles, clinical features of the patient, the anticipated side effects, and clinical trial data regarding the medication. During about 6 months following remission, the treatment that was effective in the acute phases should be used in the maintenance phase to prevent relapse.     

Targeting angiogenesis versus myogenesis with cardiac cell therapy

Considerable hope has been vested in cell therapy strategies designed to augment the endogenous neovascularization response to obstructive coronary artery disease, and to replace cardiomyocyte loss caused by myocardial infarction. Conceptually, the relative importance of targeting angiogenesis versus myogenesis in this scheme will vary depending on the clinical context (the predominance of ischemia versus ventricular dysfunction and scarring). Although the evidence so far is encouraging, whether these processes can be effectively targeted in a selective fashion with cell therapy is still unclear. Intriguingly, data are now emerging suggesting that the beneficial effects of cardiac cell therapies in a variety of clinical settings may be accounted for by a greater interaction of angiogenesis, myocardial salvage and myogenesis than heretofore appreciated, and through mechanisms that may include both cellular and paracrine effects. Greater understanding of these mechanisms should accelerate the development of effective cell therapies for the growing number of patients with advanced, and in many cases 'no-option', cardiovascular disease. Possible clinical targets for angiogenic and myogenic cardiac cell therapy, the scientific rationale for this therapeutic approach and future directions in this field are discussed here.     

Molecular target drugs for malignant lymphoma

According to revised WHO classification for lymphoid malignancies, biological differences among pathological subtypes of lymphomas could be key points for molecular target therapies. For B cell lymphomas, CD20, CD22, CD19 can be molecules for target therapies, whereas there are not so many molecular targets in T cell lymphomas yet. However, novel molecular target drugs are developing from home and abroad. Especially, inhibitors for mTOR, IKK/JAK, HDAC, proteasome, HSP90, and proapoptotic molecules are developing in clinical trials for B cell- and T cell-lymphomas, and their anti-lymphoma activities will be considerably promising. Moreover, immunomodulatory drugs such as lenalidomide are also tried to investigate the effect on lymphomas. Here, some novel molecular drugs currently under development will be reviewed about their anti-lymphoma effects.     

T-cell receptor gene transfer by lentiviral vectors in adoptive cell therapy

Adoptive cell therapy can be envisioned as a promising strategy for tumour immunotherapy. However, existing protocols of adoptive cell therapy still require optimisation as many factors, such as specificity, avidity, level of differentiation and amount of transferred T lymphocytes, can influence their immunocompetence and in vivo functionality. In particular, the need to reduce the in vitro expansion phase and to obtain large numbers of tumour-reactive T cells, as a favourable condition for cancer regression, make TCR gene transfer a potentially ideal tool to overcome the limits of adoptive cell therapy strategies. Here, the authors review the state-of-the-art and recent advances in TCR transfer with particular emphasis on lentiviral vector systems. Initial data from preclinical models and recent clinical trials encourage optimisation of a safe, simplified and stable transfer system. In this regard, HIV-based vectors are emerging as good alternative candidates over the most widely used oncoretroviral vectors due to their peculiar molecular features that fit the ideal conditions for donor T cell in vitro manipulation.     

Anaesthesia monitoring: the human factors component of technology transfer

Resistance to change in monitoring practices from within the anaesthesiology community is a formidable obstacle, and coercive and exhortatory solutions are likely to be unsuccessful in some situations. An analysis of publications about technology transfer and professional obsolescence, and application of this data to the practice of anaesthesia, reveals various stresses that technology transfer from research areas to the workplace may induce in vulnerable anaesthesiologists and account for their attitudes. It is suggested that the invaluable pronouncements of high profile anaesthesiologist groups must be supplemented by supportive behaviour by physicians and administrators at an institutional level. The human factors issues to be addressed include: (i) Monitored data acquisition skills. (ii) Possibility of acting on monitored data. (iii) Assistance for personal insight into attitudinal difficulties that may be encountered. (iv) Data supporting the value of the device. (v) Ergonomically effective integration of the monitor into the work station.Alternatively the perceptions of potential users may accurately reflect changes in their status in the new work situation created by monitors, and decision making aids that may or may not be derived from them. Thus, plans to present job satisfaction in related clinical areas or to associate the proposed new system with evaluation of its effect on patient outcome will be necessary. In this way the clinician becomes involved in clinical research, a quality of personal and quality care development.     

Gene transfer for hemophilia: can therapeutic efficacy in large animals be safely translated to patients?

Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid rather than a protein or small molecule. As early as 1997, investigators reported long-term expression of therapeutic levels of factor IX using gene transfer techniques in hemophilia B mice, and similar data were thereafter reported in mice with hemophilia A. Efforts to translate these results to hemophilic dog models at first yielded only marginally therapeutic levels (1%-2% normal circulating levels), but within the past few years have achieved levels in the range of 10%-20% through multiple different gene transfer strategies. Early phase clinical testing has revealed that many aspects of gene transfer in humans were accurately predicted by studies in hemophilic dogs, but that other aspects were not, and were only appreciated as a result of clinical testing. Studies in the next few years will determine whether the problems identified in preclinical and early phase clinical testing can be solved to develop a therapeutic gene transfer approach to hemophilia.