Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors

Gene therapy holds great promise for the treatment of a wide range of inherited and acquired disorders. The development of viral vector systems to mediate safe and long-lasting expression of therapeutic transgenes in specific target cell populations is continually advancing. Gene therapy for the nervous system is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses provide particularly attractive vehicles for delivery of therapeutic genes to treat neurological and ocular diseases, since they efficiently transduce non-dividing cells and mediate sustained transgene expression. Furthermore, novel routes of vector delivery to the nervous system have recently been elucidated and these have increased further the scope of lentiviruses for gene therapy application. Several studies have demonstrated convincing therapeutic efficacy of lentiviral-based gene therapies in animal models of severe neurological disorders and the push for progressing such vectors to the clinic is ongoing. This review describes the key features of lentiviral vectors that make them such useful tools for gene therapy to the nervous system and outlines the major breakthroughs in the potential use of such vectors for treating neurodegenerative and ocular diseases.     

Ocular gene delivery using lentiviral vectors

Substantial advances in our understanding of lentivirus lifecycles and their various constituent proteins have permitted the bioengineering of lentiviral vectors now considered safe enough for clinical trials for both lethal and non-lethal diseases. They possess distinct properties that make them particularly suitable for gene delivery in ophthalmic diseases, including high expression, consistent targeting of various post-mitotic ocular cells in vivo and a paucity of associated intraocular inflammation, all contributing to their ability to mediate efficient and stable intraocular gene transfer. In this review, the intraocular tropisms and therapeutic applications of both primate and non-primate lentiviral vectors, and how the unique features of the eye influence these, are discussed. The feasibility of therapeutic targeting using these vectors in animal models of both anterior and posterior ophthalmic disorders has been established, and has, in combination with substantial progress in enhancing lentiviral vector bio-safety over the past two decades, paved the way for the first human ophthalmic clinical trials using lentivirus-based gene transfer vectors.     

Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells

Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.     

A lentiviral strategy for highly efficient retrograde gene transfer by pseudotyping with fusion envelope glycoprotein

The lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) is used extensively in gene therapy trials of neurological and neurodegenerative diseases. Retrograde axonal transport of viral vectors offers a great advantage to the delivery of genes into neuronal cell bodies that are situated in regions distant from the injection site. Pseudotyping of HIV-1-based vectors with selective variants of rabies virus glycoprotein (RV-G) increases gene transfer via retrograde transport into the central nervous system. Because large-scale application for gene therapy trials requires high titer stocks of the vector, pseudotyping of a lentiviral vector that produces more efficient retrograde transport is needed. In the present study, we developed a novel vector system for highly efficient retrograde gene transfer by pseudotyping an HIV-1 vector with a fusion envelope glycoprotein (termed FuG-B) in which the cytoplasmic domain of RV-G was substituted by the corresponding part of vesicular stomatitis virus glycoprotein. The FuG-B pseudotype shifted the transducing property of the lentiviral vector and enhanced the retrograde transport-mediated gene transfer into different brain regions innervating the striatum with greater efficiency than that of the RV-G pseudotype in mice. In addition, injection of the FuG-B-pseudotyped vector into monkey striatum (caudate and putamen) allowed for highly efficient gene delivery into the nigrostriatal dopamine system, which is a major target for gene therapy of Parkinson's disease. Our strategy provides a powerful tool for the treatment of certain neurological and neurodegenerative diseases by promoting retrograde gene delivery via a lentiviral vector.     

Gene therapy for lysosomal storage disorders

The lysosomal storage disorders (LSD) are monogenic inborn errors of metabolism with heterogeneous pathophysiology and clinical manifestations. In the last decades, these disorders have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have allowed the successful development of bone marrow transplantation (BMT) and enzyme replacement therapy (ERT) as therapeutic options for several LSDs. However, BMT is limited by poor donor availability and high morbidity and mortality, and ERT is not a life-long cure. Moreover, the neuropathology present in many LSDs responded poorly, if at all, to these treatments. Therefore, gene therapy is an attractive therapeutic alternative. Gene therapy strategies for LSDs have employed ex vivo gene transduction of cellular targets with subsequent transplantation of the enzymatically corrected cells, or direct in vivo delivery of the viral vectors. Oncoretroviral vectors and more recently adeno associated vectors (AAV) and lentiviral vectors have been extensively tested, with some success. This review summarises the main gene therapy strategies which have been employed or are under development for both non-neurological and neuronopathic LSDs. Some of the in vitro and in vivo preclinical studies presented herein have provided the rationale for a gene therapy clinical trial for Gaucher disease Type I.     

Gene therapeutic strategies for neurodegenerative diseases

Gene transfer into the central nervous system by ex vivo or in vivo techniques is a rapidly emerging field in neuroscience. Potential applications of gene therapy for the nervous system include not only congenital single gene disorders, but also brain tumors and acquired chronic diseases. Considerable progress has been made in the understanding of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. As a result, gene therapy for Parkinson's, and possibly Alzheimer's disease could be regarded as a realistic alternative to the limited treatment options currently available. In this review, we highlight the most important developments in gene transfer techniques as well as the newest insights in the mechanisms of some neurodegenerative disorders and put these into the perspective of gene therapeutic strategies for the central nervous system.     

Lentiviral vectors for use in the central nervous system.

Lentiviral vectors have been used extensively as gene transfer tools for the central nervous system throughout the past decade since they transduce most cell types in the brain, resulting in high-level and long-term transgene expression. This review discusses some of the recent progress in this field, including preclinical gene therapy experiments in disease models, development of regulated vectors, and the application of siRNA's using lentiviral vectors. We also describe some of the features that make lentiviral vectors a likely candidate for human gene therapy in the brain.     

Cotransduction of nondividing cells using lentiviral vectors

Diseases such as AIDS and cancers may require the introduction of multiple genes into either stem cells or nondividing cells, among others, for therapeutic purposes. Such genes may act at different points of the disease pathway, or may constitute a regulatory loop to bypass or rectify the defective gene or pathway underpinning the disease. Ideally, the therapeutic genes must be transduced together in diverse combinations, and the introduction should occur without constraints. Since lentiviral vectors can transduce both dividing and nondividing cells, they are ideal vehicles to investigate combinatorial gene transfer into diverse cells. In this study, we demonstrate that by using two independent lentiviral vectors, pseudotyped with the protein g of vesicular stomatitis virus, up to four genes can be introduced simultaneously into single dividing and nondividing cells. Up to 45% and 73% of dividing and nondividing cells, respectively, could be transduced with two lentiviral vectors. The efficiency of cotransducing a single cell was the product of the individual transduction efficiencies and suggested the absence of viral interference. Multiple and combinatorial gene transduction using lentiviral vectors may prove useful in gene therapy.     

Safety features of retroviral vectors

Although retroviral vectors based upon the murine leukemia virus have good safety records from clinical trials, attention to safety issues is crucial for the advancement of retroviral gene therapy. Key issues are to reduce uncontrolled transfer of viral or non-viral genetic information and to prevent the formation of replicating retroviruses. Safety features are also being incorporated into the novel attenuated lentiviral vectors that open new prospects for gene delivery. Here, we highlight features developed to restrict the transfer of viral genes, immobilize transfer vectors in target cells, or control interactions with other retroviruses in producer or target cells, as well as new developments in tissue-targeted and regulated vectors.     

Update on gene therapy for hemoglobin disorders

The hemoglobin disorders of beta-thalassemia and sickle cell disease together constitute the most prevalent of human monogenic diseases. Although palliative therapies and curative allogeneic stem cell transplantation therapy have been developed for these disorders, many patients still suffer significant morbidity and early mortality. Therefore, development of alternative treatment based on a gene therapy approach continues to be a worthwhile endeavor. Several laboratories have recently achieved major progress towards this goal. Using lentiviral vectors to obtain high-level expression of relatively complex globin gene cassettes, therapeutic correction of several murine models of both beta-thalassemia and sickle cell disease has been achieved. These breakthroughs, coupled with recent significant developments in the ability to select and expand genetically modified stem cells in vivo, have greatly advanced the possibility of gene therapy for the hemoglobin disorders in the near future. These advances, together with recent information regarding safety issues of retroviral gene transfer, are reviewed here.     

Viral vectors for gene transfer to striated muscle

Recent progress has generated exciting results that are increasing the prospects for gene therapy of a variety of disorders of striated muscle, including the muscular dystrophies and myopathies, acquired and inherited diseases of cardiac muscle, and aging-associated muscle wasting. Numerous viral vector systems are being employed to transfer therapeutic genes to striated muscles, and advances in vector technology are leading to improved gene transfer efficiencies coupled with reduced immunological responses. The current technology in the field of viral vectors as tools for gene delivery to striated muscle is summarized, and recent developments related to gene therapies for skeletal and cardiac muscle disorders are discussed.     

Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis

Gene therapy of cystic fibrosis (CF) lung disease needs highly efficient delivery and long-lasting complementation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene into the respiratory epithelium. The development of lentiviral vectors has been a recent advance in the field of gene transfer and therapy. These integrating vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells by virtue of their ability to infect nondividing cells and mediate long-term persistence of transgene expression. Studies in human airway tissues and animal models have highlighted the possibility of achieving gene expression by lentiviral vectors, which outlasted the normal lifespan of the respiratory epithelium, indicating targeting of a 'stem cell' compartment. Modification of the paracellular permeability and pseudotyping with heterologous envelopes are the strategies currently used to overcome the paucity of specific viral receptors on the apical surface of airway epithelial cells and to reach the basolateral surface receptors. Preclinical studies on CF mice, demonstrating complementation of the CF defect, offer hope that lentivirus gene therapy can be translated into an effective treatment of CF lung disease. Besides a direct targeting of the stem/progenitor niche(s) in the CF airways, an alternative approach may envision homing of hematopoietic stem cells engineered to express the CFTR gene by lentiviral vectors. In the context of lentivirus-mediated CFTR gene transfer to the CF airways, biosafety aspects should be of primary concern.     

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.     

Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo

Modulation of adult neurogenesis may offer new therapeutic strategies for various brain disorders. In the adult mammalian brain the subventricular zone (SVZ) of the lateral ventricle is a region of continuous neurogenesis. Lentiviral vectors stably integrate into dividing and nondividing cells, in contrast to retroviral vectors, which integrate only into dividing cells. We compared their potential for gene transfer into both quiescent and slowly dividing stem cells as well as into more rapidly dividing progenitor cells. In contrast to retroviral vectors, stereotactic injection of lentiviral vectors into the SVZ of adult mice resulted in efficient and long-term marker gene expression in cells with characteristics of both immature type B cells and migrating precursor cells. After migration along the rostral migratory stream and differentiation, the number of enhanced green fluorescent protein (eGFP)-expressing granular and periglomerular interneurons increased over time in the ipsilateral olfactory bulb. Moreover, the number of eGFP-labeled neuronal progenitor cells in the SVZ increased over time. By intraventricular injection of lentiviral vectors we could restrict gene transfer to ependymal cells and type B astroglial-like stem cells. In conclusion, lentiviral vectors surpass retroviral vectors in efficient long-term in vivo marking of subventricular zone stem cells for basic research and therapeutic applications.     

Gene therapy progress and prospects: gene therapy of lysosomal storage disorders

Despite disappointments with early clinical studies, there is continued interest in the development of gene therapy for the group of metabolic diseases referred to as lysosomal storage disorders (LSDs). The LSDs are monogenic and several small and large, representative animal models of the human diseases are available. Further, the successful reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are expected to be sufficient to correct the disease at least in the case of some of the LSDs. For these reasons, they are perceived as good models for the evaluation of different gene delivery vectors and of different strategies for treating chronic genetic diseases by gene transfer. In this review, we will highlight the progress that has been made over the past 2 years in preclinical research for this group of disorders and speculate on future prospects.     

From gene transfer to gene therapy in lysosomal storage diseases affecting the central nervous system

Gene transfer into the central nervous system (CNS) is one of the foremost scientific challenges today. To give a brief survey of possible approaches to gene therapy in diseases affecting the CNS, we have selected the lysosomal storage diseases (LDS), which are an excellent model of both early-onset infantile neurological forms and late-onset adult psychiatric forms. Lysosomal storage diseases represent a group of about 50 monogenic metabolic disorders resulting from a deficiency in intralysosomal enzymes involved in macromolecule catabolism. The clinical severity, including neuropsychiatric symptoms, and the absence of an efficient therapy for the majority of these disorders prompted the various trials of gene therapy now in progress. Most of the genes encoding the normal lysosomal enzymes have been cloned, and the size of the corresponding cDNAs is generally compatible with their transfer by recombinant vectors. New vectors with improved immunogenicity, transduction efficacy, insert capacity, and specificity of targeting are under development. Here we discuss several gene therapy strategies for the correction of LSD-induced anomalies in the CNS. Interesting results have been obtained by animal model brain, which raises hopes that large-scale clinical trials may soon be started.     

Retroviral gene therapy in hematopoietic diseases

A number of diverse gene therapy strategies are being evaluated in the search for novel therapeutic approaches to hematopoietic disease. In this review, we will limit our discussion to three areas of active research: the treatment of genetic diseases, the use of drug resistance gene vectors in autologous transplantation, and tumor immunization strategies in cancer. Although gene delivery remains a major challenge to gene therapy, recent modifications which improve gene transfer will also be addressed. J. Clin. Apheresis 12:187–193, 1997. © 1997 Wiley-Liss, Inc.     

Update on hematopoietic stem cell gene transfer using non-human primate models

Gene transfer into hematopoietic stem cells (HSCs) using integrating vectors is an attractive treatment strategy for many genetic and hematological diseases. The preclinical testing of gene transfer approaches in non-human primates and other large animal models will be invaluable in order to assess toxicity and efficacy, as their HSC biology is much more closely related to humans than murine models. Gene transfer studies targeting HSCs in non-human primates have focused on optimizing gene transfer efficiency, and significant advances have been achieved using standard retroviral vectors. Utilization of lentiviral and other alternative vector system are still very preliminary in large animal models. Further development of post-transduction selection and/or expansion strategies using drug-resistance or amplifier genes will most likely be necessary for clinical applications.     

Gene therapy for the lysosomal storage disorders

The lysosomal storage disorders (LSD) are monogenic inborn errors of metabolism with heterogeneous pathophysiology and clinical manifestations. In recent decades, these disorders have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have established proof-of-concept for the development of bone marrow transplantation (BMT) and enzyme-replacement therapy (ERT) as therapeutic options for several LSDs. BMT is limited by poor donor availability and high morbidity and mortality, and although ERT is a good treatment, it is not a life-long cure. Its high cost remains an impediment for developing countries. While substrate synthesis inhibition therapy is an important idea, its clinical use is far from certain. The neuropathology present in many LSDs has responded poorly to BMT or ERT, which makes gene therapy an attractive therapeutic alternative. Oncoretroviral vectors, and more recently adeno-associated and lentiviral vectors have been tested with some success. This review summarizes the main gene therapy strategies which have been employed or are under development for both non-neurological and neuronopathic LSDs. Some of the in vitro and in vivo preclinical studies presented herein have provided the rationale for gene therapy clinical trials for Gaucher disease Type 1.