Gene Therapy for Lysosomal Storage Disorders with Neuropathology
Authors: YIANNIS A. IOANNOU
Journal: Journal of the American Society of Nephrology (2000)

Lysosomes are acidic cellular organelles that function as terminal degradative compartments (1). Biochemically, these organelles are rich in lysosomal glycoproteins, negative for mannose-6-phosphate receptors (1), and acidified through the actions of a v-type proton ATPase (2). More than 40 acidic hydrolases reside in the lumen of this organelle and catalyze the stepwise degradation of complex carbohydrate, protein, and lipid substrates (3). Lysosomal storage diseases (LSD) are a group of approximately three dozen heterogeneous human disorders characterized by the accumulation of undigested macromolecules within the lysosomes (Table 1), resulting in an increase in the size and number of these organelles (4). In 1965, Hers (5) developed the concept of LSD to explain the relationship between α-glucosidase and Pompe disease. Initially, it was ambiguous whether LSD arose by lack of degradation or by increased synthesis of the accumulated substrates; however, experimentally induced lysosomal storage of compounds such as sucrose and dextran seemed to suggest the former alternative (6). LSD are generally classified by the accumulated substrate (7), and they include sphingolipidoses, glycoproteinoses, mucolipidoses, mucopolysaccharidoses, and others. It is now known that the LSD result from a deficiency of a specific lysosomal enzyme or protein; however, it is still unclear how storage of accumulated substrates relates to the pathology seen in many of these disorders.Table 1: The lysosomal storage disordersaWith a few exceptions, these disorders lead to a severe neuro-degenerative phenotype (Table 1), which further complicates potential treatment modalities, including gene therapy. In some diseases (e.g., Gaucher, Niemann-Pick, Tay-Sachs, and Schindler), milder or late-onset subtypes have been identified, owing to the presence of residual enzymatic activity. These subtypes and variants demonstrate that even low levels (1 to 5% of normal) of enzyme activity can alter a severe neurodegenerative disease course to a milder, often non-neurologic phenotype. It should be noted, however, that these low residual activities are present in all cells or at least in those that require these enzymes. This is an important point that must be taken into consideration when developing enzyme or gene therapy protocols. Thus, replacing 1 to 5% of the circulating enzyme in these disorders may not be sufficient to revert the disease phenotype to that seen in the milder forms. Lysosomal Storage Diseases as Therapeutic Models For more than three decades, these disorders have been models for the development of therapeutic endeavors for inherited metabolic diseases. In the early 1970s, it was shown that low levels of the proper normal enzyme could correct the metabolic defect in cultured fibroblasts from affected patients with various types of LSD (8,9,10). This observation fascinated investigators, who have since pursued enzyme replacement as a means of treating these disorders. For example, clinical trials of enzyme replacement in patients with type 1 Gaucher disease have confirmed the effectiveness of this approach in reversing the reticuloendothelial cell pathology in affected individuals (11). These long-term clinical trials provide dramatic evidence of the clinical effectiveness of enzyme replacement for a non-neuronopathic lysosomal disease. However, enzyme replacement trials in patients with the neuronopathic forms of Gaucher disease (types 2 and 3) have been unsuccessful, attesting to the inability of an intravenously administered enzyme to cross the blood-brain barrier (BBB) (12). Bone marrow transplantation (BMT) studies (13) yielded analogous results. Most BMT recipients do not experience neurologic or intellectual improvement, and most have a prolonged clinical course before experiencing a neurologic demise. These studies reflect the present limitations of BMT for the treatment of LSD with significant neurologic involvement and emphasize the need to develop novel strategies to treat LSD whose primary site of pathology is the central nervous system (CNS). Clearly, the LSD have been invaluable paradigms for the development of novel therapeutic strategies such as enzyme replacement therapy (ERT) and will continue to serve as excellent model systems in which to develop and evaluate new disease treatments. Lessons from Enzyme Replacement in Animal Models ERT for several LSD has been reported using a variety of animal models. For example, MPS type VII mice receiving single or multiple injections of β-glucuronidase exhibit attributable substrate reduction in liver and spleen, whereas reduction in heart and kidney is seen only after multiple injections (14). MPS type I dogs receiving multiple injections of α-Liduronidase show substantial decreases in storage in liver, spleen, and kidney, but none in heart (15, 16). At low doses, only liver Kupffer cells are cleared, whereas at high doses, both Kupffer cells and hepatocytes are cleared (16). MPS type VI cats administered multiple injections of N-acetylgalactosamine-4-sulfatase show reversal of lysosomal accumulation in liver and heart (17), but heart depletion is dependent on dose (18). Early studies of ERT in humans with LSD also provide evidence of catabolism of accumulated substrates in tissues of patients. For example, some patients with glycogenosis type II administered human or Aspergillus niger α-glucosidase show decreases in glycogen accumulation in liver (19). Also, purified human acid β-glucosidase is capable of reducing storage in liver, red blood cells, lymphocytes, and platelets in patients with Gaucher disease (20). And most importantly, urinary globoside concentration is significantly decreased in a patient with Sandhoff disease following infusion of plasma, suggesting a catabolism of renal glycolipid by active hexosaminidases (21). Finally, as mentioned above, the most successful example of ERT is that for Gaucher disease type I (22). The administration of recombinant β-glucosidase in this disease has been an effective mode of treatment for almost 10 years (reviewed in reference (23). The results of these studies, albeit mixed, provide important clues regarding the proper dose and mode of delivery of a therapeutic protein. Clearly, replacing a deficient lysosomal enzyme requires a large dose of recombinant protein; this has major implications for gene replacement endeavors. Gene Therapy for LSD Because ERT is clearly limited to disorders without CNS involvement, other modalities for treatment must be explored for diseases with neural involvement. One of the most promising approaches involves gene delivery to the brain. However, the brain presents unique physical and logistical barriers to most, if not all, gene therapy approaches. Also, it should be emphasized that the neuropathology in LSD is spread throughout the entire brain and is not limited to specific areas, thus requiring global neural gene delivery for effective treatment. Thus, the development of novel strategies to deliver genes to neurons and other neural cells throughout the CNS is necessary to achieve the continuous expression of the proper therapeutic enzyme and correction of the metabolic defect. The main obstacles to this goal are the difficulty in delivering genes to the CNS, and, within the CNS, the limited accessibility of neurons, the main site of neuropathology in the LSD, to the therapeutic gene. To overcome these obstacles, novel “global neural delivery” and “neural cell targeting” strategies must be developed. Early efforts to solve the above-mentioned problems involved using viral vectors to deliver genes directly into the CNS and were of limited success. For example, herpes virus vectors have been used to express the lysosomal enzyme β-glucuronidase in the brains of mice following stereotactic injection. However, very few cells were transduced and they remained clustered near the injection tract (24). Furthermore, issues of cytotoxicity remain a problem with herpes virus vectors. Adenovirus vectors have also been stereotactically introduced into the brain and have been used to express various marker proteins. However, analogous to the results obtained with the herpes virus vectors, the percentage of cells that was transduced in vivo was very low and the cells did not appear to migrate significantly from the site of injection (reviewed in reference (25). In addition, questions remain regarding the persistence of expression in various neural cells using viral vectors. Alternatively, nonviral delivery systems have been used to express genes in the brains of animals, but these also have had limited success (reviewed in reference (26). Thus, little progress has been made regarding the in vivo delivery of genes to the brain and the proper expression of these genes in neural cells. Moreover, efforts to deliver genes to the entire brain (i.e., global CNS delivery) have lagged behind, mostly because of the presence of the BBB and the failure of intravenously administered gene delivery vehicles to cross this barrier. An approach with a high potential for achieving global neural gene delivery involves the transient disruption of the BBB and injection of nonviral DNA delivery vehicles. Although the transient disruption of the BBB has been carried out successfully both in animals (27) and in humans for the delivery of chemotherapeutic agents to combat brain tumors (28), delivery of gene packages has not been attempted until now. We have made a number of assumptions to devise a scheme for effective delivery of genes to the CNS. First, polycationic polypeptides that can complex and compact the plasmid DNA are available. Second, these compacted DNA packages can be targeted via specific cell ligands for plasma membrane binding and initial internalization. Third, endocytosed DNA packages can be designed to bypass the endosomal/lysosomal system. Fourth, nuclear localization signals in these packages will ensure efficient nuclear localization. We propose that all of the above prerequisites can be met by the use of in vivo selected peptide sequences fused to the small, DNA-compacting protein protamine. The following discussion describes our approach to formulating these DNA packages, encoding the Aequorea victoria green fluorescence protein (GFP), which can cross the BBB and transduce neural cells. DNA Compaction An important consideration, for crossing the BBB in particular and for any nonviral gene delivery vehicle in general, is the size of the DNA complex. Recent work on poly-L-lysine (PLL) as a condensing moiety for DNA has identified conditions by which the size of the PLL-DNA complex can be controlled and reduced to approximately 20 nm, compatible not only with endocytic uptake (limit approximately 100 nm), but also for diffusion through various tissues in vivo (see below). Furthermore, even smaller DNA particles can be procured by using PLL of lower molecular weight (approximately 3.5 kD), which has the added advantage of reduced toxicity. It has also been shown that derivatization of PLL with appropriate glycans improves cellular transfection and transduction, presumably attributable to a more efficient uptake of the complex (29) and/or decreased toxicity. Finally, PLL is easily derivatized biochemically and thus can provide extremely useful experimental data relevant to the development of the conjugate artificial vector that will eventually be obtained by recombinant DNA techniques. Alternatively, salmon protamine (SP), a protein found naturally complexed with DNA at high concentrations in spermatozoa, can be used. This small 4-kD arginine-rich cationic protein can be engineered as a fusion protein to any cell-specific peptide (see below), resulting in a cell-targeted chimeric protein that can bind to the expression construct DNA. To obtain complexes between pGL, a 5-kb plasmid encoding GFP driven by the cytomegalovirus promoter, and either of the DNA-compacting proteins, PLL (approximately 22.5 kD) or SP (approximately 4 kD), we used a published procedure with appropriate modifications (30). By tightly controlling the DNA:protein ratio, reaction volume, timing, order of addition, and salt concentration, we have consistently obtained apparently torroidal PLL-DNA and SP-DNA particles of approximately 10 to 20 nm in diameter, as assessed by transmission electron microscopy (TEM) (Figure 1). Preliminary TEM evidence suggests that by using PLL 3.5 kD, the diameter of the torroidal complexes can be further reduced to <10 nm, which should facilitate intercellular movement.Figure 1.: Electron microscopy of compacted DNA. Transmission electron micrographs of poly-L-lysine-DNA (A) and salmon protamine-DNA (B) complexes. Apparently torroidal particles 10 to 20 nm in diameter are obtained. Bar, 100 nm.Cell-Specific Targeting and Endosomal Escape The choice of targeting ligand depends on the cell type to be targeted and would preferably be a peptide recognized by a receptor (not necessarily a protein) that undergoes endocytosis. Although most endocytosed plasma membrane proteins/receptors enter the endosomal/lysosomal system, recent evidence suggests that an alternate nonendosomal uptake system is functional in most cells (31). Support for the existence of this uptake system has recently been generated through studies of the small DNA virus, SV40, which apparently enters cells in an endosome-independent manner involving noncoated, caveolae-like vesicles (32). In addition to SV40, several receptors are internalized via this system (reviewed in reference (33), making it an attractive targeting pathway that can bypass the problems and limitations associated with entering clathrin-coated endosomes. The recent availability of phage peptide display libraries allows the rapid selection of small peptides with specific binding properties. Phage display libraries have been used in several in vitro selection procedures to isolate ligands that bind to specific proteins (34) and recently in an in vivo system to isolate peptides that bind to specific mouse organs (35). Using A431 cells, a commercially available, seven amino acid Escherichia coli phage display library was used to determine whether small peptides that undergo internalization via an endosome-independent pathway could be isolated. Cells were plated in culture dishes and the phage library, representing about 5 × 109 independent phage particles, was allowed to adsorb onto the cell monolayer. The rationale for this experimental setup was the following: Phage that were endocytosed via an endosome-dependent pathway would be partially or completely destroyed in the lysosomal system, whereas phage that were internalized by an endosome-independent pathway would be left intact and could be recovered by infecting fresh Escherichia coli. The cells were subsequently washed to remove any phage particles that had not been internalized, lysed, and used to infect competent Escherichia coli cells to recover viable phage particles. Recovered phage were expanded en masse and used for another round of adsorption onto A431 cells as above. This procedure was repeated three times to reduce or eliminate background, and the phage recovered after the third round were sequenced to determine the identity of the displayed peptides (Figure 2). The peptides identified by this procedure (out of a pool of > 109 particles) show a consensus sequence in 5 of 10 sequences. The proline at position 4 (Figure 2) suggests that these phage particles were endocytosed and escaped endosomal degradation by an analogous mechanism, probably via the same receptor system. This consensus, hydrophobic-X-Pro-X-positively charged (Gln or Asn), may be important in endocytosis and endosome escape. Furthermore, analysis of peptides capable of binding to MHC class I receptors reveals a striking similarity between the isolated peptides and MHC binding peptides, which also contain proline and glycine in similar positions.Figure 2.: Peptides isolated by in vivo selection. A ClustalW alignment of 10 peptides isolated by in vivo selection as described in the text. The conserved proline residues are boxed. A consensus sequence of hydrophobic-X-proline-charged amino acid can be clearly seen in the isolated peptide sequences.Thus, in vivo screening of phage display libraries can lead to the isolation of small peptides with novel and desirable cell targeting and uptake characteristics. Such screening procedures can easily be adapted to the isolation of peptides taken up by specific cell types, such as neuronal cells, by an endosome-independent pathway for delivery of DNA constructs to the nucleus. Nuclear Localization Once the vector DNA complex is released into the cytosol of the targeted cells, the DNA must gain access to the cell nucleus. Nuclear targeting can be achieved by incorporation of a nuclear localization signal into the DNA-containing complex. Such signals have been well characterized for several mammalian and viral proteins and appear to be small, positively charged peptides of about 7 to 10 residues. Nuclear targeting is particularly important for cells that do not actively divide. However, use of PLL or protamine to formulate the DNA complex may obviate the need for a nuclear localization signal, because these polycations are already effectively targeted to the nucleus. Global Neural Delivery A method to bypass the BBB is necessary to access the CNS in a global manner. The extensive vascularization of the brain provides an attractive delivery route. However, the BBB prevents the passage of macromolecules from the circulation to the CNS parenchyma. An approach that can be used to deliver DNA packages to the brain is the transient opening of the BBB by the intracarotid injection of hyperosmolar solutions of mannitol or other agents (36). Electron microscopic observations have confirmed that hyperosmolar solutions cause dehydration and shrinkage of vascular endothelial cells, thereby transiently relaxing the tight junctions and opening the intercellular spaces (reviewed in reference (37). This method provides the means to globally deliver relatively large DNA packages (10 to 20 nm) to the brain. In humans, hyperosmolar BBB disruption has been applied to patients for the treatment of certain brain tumors (38). The therapeutic effectiveness of the procedure has been limited due to its cytotoxicity to normal cells (39), resulting from the high concentration of cytotoxic drugs delivered to the entire CNS parenchyma. These observations attest to the effectiveness of this strategy as a global CNS delivery system. Moreover, few complications have been reported in patients (40) or animals undergoing short-term experiments (41), thereby justifying its use to deliver nontoxic, therapeutic DNA vector constructs to the brain parenchyma. To test this CNS delivery approach, protein-DNA complexes were infused by intracarotid injection into rats after BBB disruption. Neural cell transduction by uptake of protein-DNA packages was judged by GFP expression in rats, which were sacrificed 5 d after treatment and examined by confocal microscopy. Numerous GFP-positive parenchymal cells were detected in the infused animals (Figure 3). In addition, positive neural cells can be seen expressing GFP, suggesting that the compacted DNA packages can be endocytosed by all neural cells. Thus, the transient disruption of the BBB and infusion of compacted DNA packages can accomplish the initial requirement of global neural delivery of therapeutic genes to the brain.Figure 3.: Confocal microscopy of rat brain sections. Micrograph of rat brain section showing green fluorescence-positive cells (arrows) due to expression of the green fluorescence protein (GFP) transgene in numerous parenchymal cells. (A) Phase contrast of GFP field shown in Panel B.Conclusion In conclusion, ERT has not proved to be a viable mode for treatment of LSD with neuropathology. Gene therapy approaches using current viral vectors have also been ineffective due to the presence of the BBB. Based on our experimental data, delivery of nonviral DNA complexes to the brain after transient disruption of the BBB appears to be the most promising approach to global neural delivery of therapeutic genes. Acknowledgments I acknowledge the contribution of many people in my laboratory, first and foremost that of Dr. Mario Rattazzi, whose expertise in BBB disruption has made this work possible, and Ronald Gordon for electron microscopy. In addition, I thank Compton Benjamin, Takashi Tsuda, and Annette Enriquez for their hard work. Finally, I thank Fannie W. Chen for careful review of the manuscript.