Gene therapy approaches for neurological disorders

 Gene Therapy Approaches for Neurological Disorders (CNS): progress and prospects

Abstract

Gene therapy for neurological disorders is currently an experimental concept. The goals for clinical utilization are the relief of symptoms, slowing of disease progression, and correction of genetic abnormalities. Experimental studies are realizing these goals in the development of gene therapies in animal models. Discoveries of the molecular basis of neurological disease and advances in gene transfer systems have allowed focal and global delivery of therapeutic genes for a wide variety of CNS disorders. Limitations are still apparent, such as stability and regulation of transgenic expression, and the safety of both vector and expressed transgene. In addition, the brain adds several challenges not seen in peripheral gene therapy paradigms, such as post-mitotic cells, heterogeneity of cell types and circuits, and limited access. Moreover, it is likely that several modes of gene delivery will be necessary for successful gene therapies of the CNS. Collaborative efforts between clinicians and basic researchers will likely yield effective gene therapy in the CNS.

Introduction

Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood-brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this is a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors.

Exogenous and endogenously regulated gene therapies are each associated with a set of advantages and disadvantages when considering their use in patients. Firstly, exogenously regulated systems usually require long-term administration of a drug. This necessitates extensive characterization of the safety and tolerability of the drug, which, with the exception of tetracycline, has not occurred thus far. Additionally, it is not possible to tailor gene expression precisely to pathological signals using exogenous systems unless they are associated with a noticeable phenotypic change in a timely manner. In most cases, there is a latent period between the presence of a pathological signal and the appearance of clinical symptoms. Therefore, endogenous systems have the advantage of responding in a timelier manner and with an intensity that is tailored to the individual patient.

Additionally, gene expression systems are typically administered to whole organs or entire regions of organs, and while it may be advantageous to express the transgene in this manner; this is quite often not the case. By linking transgene expression to a pathological signal, the recombinant protein will only be produced in cells that require it, and toxic overexpression in healthy cells can be avoided. Conversely, a major limitation of endogenous systems is that once administered it will be virtually impossible to stop transgene expression in the event of an adverse reaction, unless the population of cells of interest is removed, or there is a built-in safety mechanism. Additionally, the pathological signals used need to be as far upstream as possible and be specific to the disease phenotype to enable transgene to be expressed in a timely and precise manner.

Genetic therapy covers a range of methods for modifying the nervous system, including the delivery of genes, sequence-targeted regulatory molecules, genetically modified cells and oligonucleotides. In this review, we focus on ways to change the genetic status of the nervous system, including routes of access and modes of delivery. Examples are provided of strategies used for a number of diseases, including recessive loss of function conditions [lysosomal storage diseases and spinal muscular atrophy (SMA)], dominant toxic mutations [amyotrophic lateral sclerosis (ALS)] and conditions of mixed etiology [Alzheimer's disease (AD), Parkinson's disease (PD) and brain tumors] Neurodegenerative diseases caused by triplet nucleotide repeats are discussed in an article on siRNAs in this issue. Many of these strategies have proven very promising in preclinical studies in mouse and larger animal models, and a substantial number are now being evaluated in clinical trials.

 

Therapeutic strategies based on genetic etiology. (A) Recessive disease. In the case of a recessively inherited disease where both alleles (and one on the X chromosome in males) of a gene are mutated, the goal is to replace the defective gene with a functional. Traditional pharmacological approaches often run into considerable challenges when treating CNS disorders. It is difficult to get many compounds across the blood-brain barrier (BBB). Even for compounds that cross the BBB, very large doses must be administered into the blood to get enough of the drug into the brain to be effective. This can often lead to side effects in peripheral organs that must be considered. Methods to concentrate the drug within the nervous system, such as intrathecal administration are possible but chronic administration of the drug has a significant risk of complications.

 

The benefit of gene transfer is that the therapeutic agent (protein, siRNA, etc.) can be produced within the CNS and provided on a permanent steady-state basis after a single administration. AAV vector technology is advancing rapidly, facilitating new approaches for clinically relevant gene transfer. Changes to the capsid, genome design, and route of administration have made global CNS gene transfer possible in ways that are expected to translate to humans. This has led to a renaissance of sorts in gene therapy strategies to treat inherited neurological disorders.

 

Recent years have seen a plethora of potential gene therapy strategies for the central nervous system (CNS) disorders. One of the major challenges gene therapy applications face clinically is the ability to control the level of expression or silencing of therapeutic genes in order to provide a balance between therapeutic efficacy and nonspecific toxicity due to overexpression of therapeutic protein or RNA interference-based sequences. Thus, the ability to regulate gene expression is essential as it reduces the likelihood of potentially initiating adverse events in patients. Although genes maybe regulated at either the translational or posttranscriptional level, greatest success in gene regulation has been at the transcriptional level and as such gene regulation systems at a transcriptional level are the focus of this review.

 

There are two classes of gene regulation systems—exogenously controlled gene regulation systems, which rely on an external factor (usually the administration of a drug) to turn transgene the expression on or off, and endogenously controlled gene expression systems that rely on physiological stimuli to control transgene expression. This review covers the characteristics of an ideal regulatory system and summaries the mechanics of current gene regulation systems and their application to CNS disorders

 

Related work discussion

The concept of gene delivery using virus-derived vectors was introduced in the mid-twentieth century. In this chapter, we review the different viral-based vectors (Retro-, Lenti-, Adeno- and Adeno associated viruses) developed over this time in clinical trials for treating different CNS pathologies including brain tumors, Parkinson’s disease, Alzheimer’s disease, and cancer pain. We describe the successes achieved and the challenges still faced for each of these viral vectors as well as brain disease conditions. This approach was widely perceived as ground-breaking for treating a wide spectrum of genetic diseases (Friedmann et al. 1976). The development of efficient viral vector platforms rapidly propelled human gene therapy to the forefront as a means to correct otherwise fatal disorders. Simple retroviral vectors (γ-retroviruses) were among the first that were utilized in preclinical and clinical studies due to low immunogenicity, long-term transgene expression, and a relatively simple manufacturing protocol.

 

The first proof-of-principle study using γ-retroviruses to correct a genetic disease in humans was a trial attempting to correct a severe combined immunodeficiency disorder (SCID) carried out in Kenneth Culver's laboratory (Blaese et al.1995). In this clinical trial, CD34-positive cells were isolated from two patients with inherited adenosine deaminase deficiency, transduced ex vivo with a γ-retrovirus, which carried the normal version of the gene, and readministered to the patients. One patient exhibited a temporary response, although she continued on enzyme replacement therapy (ERT). The response was far more limited in the second patient. Similar clinical trials were later conducted by Alain Fischer's group in France (Cavazzana-Calvo et al. 2000). (Gaspar et al. 2004).

 

Tragically, in both clinical trials, several children developed T-cell leukemia within 2–5 years after gene therapy, and one of these children died. Analysis of the patients with leukemia revealed intentional mutagenesis in the leukemic T-cell clone, which was correlated with the onset of leukemia. Integration of the provirus resulted in up-regulation of adjacent proto-ontogenesis due to the strong promoter elements in the γ-retrovirus long terminal repeats (LTR). Until this incidence, the risk of inspectional mutagenesis of retroviruses was estimated to be only 10−6–10−8 per integration event (Stocking et al., 1993). Currently, the frequency for a transforming insertion in a region of 10 kb around a proto-oncogene is calculated as 10−2–10−3 (Baum et al. 2003). Which highlights the limitations of the γ-retroviral vector approach.

 

HSV-1 recombinant virus and amplicon vectors Herpes simplex virus type 1 (HSV) is a common pathogen in humans, causing primarily cold sores, but occasionally encephalitis and other life-threatening conditions, especially in immune-compromised individuals. It is an enveloped virus bearing 152 kb of double-stranded DNA encoding over 80 genes, which has high infectivity for neurons and glia, as well as many other cell types.

 

The virion enters the cell by fusion of the envelope with the plasma membrane, and the capsid is transported along microtubules to the nucleus. In neurons, HSV vectors are delivered by rapid retrograde transport along neurites to the cell body, providing a means of targeting gene transfer to cells that are difficult to reach directly. The viral DNA is deposited in the nucleus, initially in a circularized episomal form, and eventually replicates, enters latency or is degraded depending on its composition. Two types of vectors are derived from HSV: recombinant virus vectors (RV) and amplicon vectors (Glorioso et al. 2007).HSV-RV vectors contain the full viral genome mutated in one or more virus genes to reduce toxicity and provide space for transgenes (30–50 kb).

 

Replication-conditional RV vectors can selectively replicate in and kill tumor cells in the brain. Replication-defective RV vectors are designed to have minimal toxicity, and current versions delete multiple immediate–early genes that encode transactivating factors, thereby essentially eliminating expression of other viral genes, eg deletions of genes encoding ICP4, ICP22, ICP27 and ICP47 (the latter being involved in antigen presentation). Elimination of ICP0 further reduces toxicity in some cells but also results in low levels of transgene expression.RV vectors can enter a stable, benign, episomal latent state in neurons, but with consequent down-regulation of most viral and cellular promoters. The long-term expression has been achieved in neurons using the LAT promoter(s) which are active in viral latency. The HSV amplicon vector consists of a plasmid bearing the HSV origin of DNA replication, oris, and packaging signal, pac, which allows it to be packaged as a concatenate in HSV virions in the presence of HSV helper functions. These vectors can be packaged free of helper HSV virus by cotransfection with the HSV genome deleted for space signals using a set of cosmids or BAC plasmid.

 

The likelihood of insertional mutagenesis might be lower when utilizing lentiviral vectors. For example, in a model with tumor-susceptible mice, transplantation of γ-retroviral vector-transduced hematopoietic cells resulted in an accelerated tumorigenic process, whereas no additional adverse events were detected with lentiviral vectors (Montini et al. 2006). Furthermore, it has been demonstrated that a higher quantity of lentiviral vectors is necessary to cause an oncogenic risk similar to that of γ-retroviral vectors (Montini et al. 2009). Thus, the use of lentiviral vectors should provide significant advantages in reducing the potential for adverse mutagenic events. Interestingly, this problem had already been taken into consideration from a theoretical point of view before the clinical trials mentioned above (Cline et al.1985) (Hacein et al.2003)(Hacein-Bey-Abina et al. 2003).

 

Another major disadvantage of using γ-retroviral vectors is the fact that they only transduce dividing cells. The infection of nondividing cells is possible, but the nuclear membrane must be disassembled for the integration of the viral cDNA into the host cell genome (Lewis et al.1994). (Miller,et al.1990). Thus, in order to target nondividing or terminally differentiated cells (e.g., post-mitotic neurons) lentiviral vectors should be employed. Nuclear import of the lentiviral genome is maintained by the host proteins (Lewis et al. 1994). Efficient transduction of neuronal cells in vivo was shown in the very first publication that utilized a lentiviral platform for the gene delivery (Naldini et al. 1996). Lentiviral vectors also have been shown to transduce most cell types within the CNS in vitro and in vivo, including premitotic and postmitotic neurons, adult neuronal stem cells, astrocytes, and oligodendrocytes (Blomer et al. 1997)( Consiglio et al. 2004).

 

The first clinical trials that employed lentiviral vectors to treat inherited disorders for adrenoleukodystrophy (ALD) (Phase I/II) (Cartier et al., 2009) and β-thalassemia (currently Phase III) were conducted in Europe. These clinical trials provided evidence of therapeutic efficacy in several patients for at least 6 years. Another clinical trial utilized lentiviral vectors for the delivery of multiple genes involved in dopamine biosynthesis. This vector (ProSavin) is currently being tested in Phase I/II trials for PD (Grosset, 2010)( Stewart et al. 2011). Finally, two more recent trials utilized lentiviral vectors for gene therapy of inherited diseases; metachromatic leukodystrophy (MLD) (Biffi et al., 2013) (Phase I/II), and Wiskott–Aldrich syndrome (WAS) (Aiuti et al. 2013) (Phase I/II) are discussed in this chapter.

 

CONCLUSION

Promising results have been obtained in small clinical trials for MLD and WAS using lentiviral vectors by ex vivo gene transfer approaches, but a clear understanding of the extent of rescue and confirmation of these preliminary results will require larger clinical trials. Early trials also identified the risk of insertional mutagenesis, ultimately leading to oncogenesis. Emerging technology for lentiviral vectors appears to overcome many of the early safety concerns for retroviral vectors.

For AAV vectors, early clinical trials demonstrated successfully gene transfer, but large impacts on the disease symptoms were lacking except perhaps for the small AADC-deficiency trial. Clinical trials for PD have shown encouraging results, but are plagued by a placebo effect that has made assessing the potential of gene therapy difficult. LSDs represent a promising family of diseases that could benefit from gene therapy. The main obstacle in the translation of LSD gene therapies has been the availability of a global gene delivery system applicable to large animals; however, promising technological developments utilizing IV or intra-CSF AAV vector delivery are beginning to meet that need.

In summary, the clinical trials to date have laid important groundwork in the advancement of CNS-directed gene therapy. While an unequivocal clinical success for CNS gene therapy has remained elusive, upcoming clinical trials will be testing approaches that have much greater potential to successfully translate encouraging results from animal models into humans. In the upcoming years, clinical trials for SMA, GAN, MPS IIIB, and other diseases will test the promise of global AAV-mediated CNS gene transfer. Concurrently, the promising results from the MLD and WAS trials using lentiviral vectors are being translated to other related diseases. Once a gene therapy breakthrough is realized for one disease, no matter how rare, the vector platform and approach can serve as a template for the treatment of a wide range of neurological disorders.

 

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